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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
  • C12N5/0623—Stem cells
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/12—Chemical aspects of preservation
  • A01N1/122—Preservation or perfusion media
  • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
  • A61D19/00—Instruments or methods for reproduction or fertilisation
  • A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/11—Epidermal growth factor [EGF]
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/52—Fibronectin; Laminin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/54—Collagen; Gelatin

Definitions

• the present invention relates to an improved method for vitrification of biological cells, especially blastocyst-derived stem cells (BS cells).
• BS cells blastocyst-derived stem cells
• a stem cell is a cell type that has a unique capacity to renew itself and to give rise to specialized or differentiated cells. Although most cells of the body, such as heart cells or skin cells, are committed to conduct a specific function, a stem cell is uncommitted, until it receives a signal to develop into a specialized cell type. What makes the stem cells unique is their proliferative capacity, combined with their ability to become specialized. For years, researchers have focused on finding ways to use stem cells to replace cells and tissues that are damaged or diseased. So far, most research has focused on two types of stem cells, embryonic and somatic stem cells. Embryonic stem cells are derived from the preimplanted fertilized oocyte, i.e.
• blastocyst whereas the somatic stem cells are present in the adult organism, e.g. within the bone marrow, epidermis and intestine.
• a fertilized oocyte is not regarded as an embryo before implantation in the uterus i.e. 10-14 days after fertilization, and such cells are therefore referred to as blastocyst-derived stem cells or hBS cells herein when employed according to the invention.
• Pluripotency tests have shown that whereas the embryonic or blastocyst-derived stem cells can give rise to all cells in the organism, including the germ cells, somatic stem cells have a more limited repertoire in descendent cell types.
• vitrification a sample containing the cells is rapidly cooled down to very low temperature and then the water content forms a glass-like state without crystallizing. Thus; vitrification is rapid cooling of a liquid medium in the absence of ice crystal formation.
• An amorphous glass forms during rapid cooling by direct submission into liquid nitrogen of e.g. a straw containing the cells. The glass retains the normal distribution of the liquid but remains in a supercooled form.
• vitrification is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth.
• the present invention relates to a method for vitrification of cells, comprising
• the present invention relates to a method for vitrification of cells in closed straws with dimensions that allow a volume from about 20 ⁇ l to about 250 ⁇ l, such as, e.g., from about 20 ⁇ l to about 225 ⁇ l, from about 25 ⁇ l to about to about 200 ⁇ l, from about 25 ⁇ l to about 175 ⁇ l, from about 25 ⁇ l to about 150 ⁇ l, from about 30 ⁇ l to about 125 ⁇ l, from about 30 ⁇ l to about 100 ⁇ l, from about 35 ⁇ l to about 75 ⁇ l, from about 40 to about 50 ⁇ l to be contained in them.
• the straws used in the provided examples of the present invention are approximately 13 cm long, a diameter of about 2 mm and a very thin plastic wall of about 0.1 mm (closed straws, French mini-straws, 250 ⁇ l, L'Aigle, IMV ZA 475°, 133 mm, Svensk Mj ⁇ lk).
• closed straws French mini-straws, 250 ⁇ l, L'Aigle, IMV ZA 475°, 133 mm, Svensk Mj ⁇ lk.
• the dimensions of the container with liquid nitrogen allows the entire length of the straw to be covered by liquid nitrogen.
• closed straw Another very important step in the above-mentioned method is the use of so-called closed straw.
• closed straw is used to denote straws that in the filling position have an open end to enable filling with the biological material (e.g. the cells or cell lines) e.g. in a suitable medium, but this end is immediately after filling tightly closed to avoid unwanted contamination of the cells from the surroundings and also to avoid the risk of unwanted contamination of the surroundings from the cells. Airtight seals on both ends of the straw are important to prevent contamination of both the samples and the environment.
• a suitable system is a Manual Sealing Unit called CBS SYMS from Cryo Bio System.
• the straws are open from one side and have a stopper in the other side.
• This stopper allows air to be sucked with a syringe in order to fill the straw with liquids, but polymerizes once it gets in direct contact with a liquid, sealing the capillary at this end. Other suitable ways of sealing this end may also be applied.
• the other end will then be closed using a sealing (weld, bond, or the like).
• the wall is thin and the diameter is small which allows for rapid cooling of the content in the straw.
• the length is not so critical but for practical reasons it is good that is of standard length so it fits in standard holders in a liquid nitrogen tank.
• the straw is made of plastic but can be made of any suitable material including glass (although this might break easier). Important is that the material is safe and no substances can be absorbed or released, that it is non-porous, non-toxic, and biocompatible.
• cryopreservation denotes the preservation of biological material at an extremely low temperature.
• directly contacted or “directly exposed” used in the present context mean that a biological material is “directly contacted” or “directly exposed” to e.g. a freezing material if a surface of the biological material or the medium, solution or material in which the material resides is allowed to come into contact with the freezing material.
• freezes material denotes any material that is capable of causing vitrification of a biological material.
• any freezing media that is cold enough can be used since the samples are not in direct contact with it.
• Suitable materials include, but are not limited to, liquid gasses like liquid nitrogen, liquid propane, liquid helium, ethane or the like.
• “Viable” used herein means that a biological material is able to live, develop and proliferate normally for a period of time.
• a volume of at least 20 ⁇ l biological material e.g. hBS cells or hBS cell lines
• a freezing material suitable preferably liquid nitrogen.
• the cells undergo vitrification and can then be stored for a period of time and thawed at a later date.
• the thawed biological material remains viable.
• there is no direct contact or direct exposure of the cells and the freezing material there is no direct contact or direct exposure of the cells and the freezing material. Thus, the risk for contamination of the cells (from external sources) as well as contamination of the environment with the cells is avoided.
• the biological material of the present invention are living cells or cell lines especially BS cells, BS cells or cells derived from BS cells.
• the cells may be in any stage of development.
• the cells are derived from an animal source including a mammalian source including, but not limited to humans, non-human primates such as monkeys, laboratory animal such as rats, mice and hamsters, agricultural livestock such as pigs, sheep, cows, goats and horses.
• the cells are human stem cells including human BS cells.
• Suitable cells for use in a method according to the invention are BS cells or BS cell lines, especially hBS cells or hBS cell lines.
• the cells or cell lines may be obtained using the procedure described herein.
• At least one of the vitrification solutions may contain one or more cryoprotectants or mixtures of cryoprotectants.
• Non-toxic cryoprotectants are of course preferable.
• Cryoprotectants help minimizing shrinking by reducing the mole fraction of other solutes remaining in the non-frozen water. They inhibit the formation of crystalline ice, and thus depress the freezing point of the water. They may also prevent protein denaturation by hydrogen binding with bound water. As cells cool, solvent water converts to extracellular ice, and the increasing extracellular concentration of non- permeating electrolyte or non-electrolytes damages the cells. When treated with a cryoprotectant, cells do not reach the salt concentrations of non-treated cells until they reach much lower temperatures.
• cryoprotectants may also function as osmotically active agents.
• Suitable cryoprotectants can be selected from the group consisting of ethylene glycol, propylene glycol, dimethylsulfoxid, glycerol, propane diol, sugars including sucrose, trehalose, maltose, lactose and methyl pentane diol.
• concentration of the individual agents contained in the first and or the second solution is normally in a range of 5-50% v/v such as, e.g. from about 5% to about 40% v/v such as e.g.
• the total concentration (i.e. calculated as v/v, w/v or M) of the cryoprotectant in the second solution is larger than that in the first solution.
• the first and the second solution may contain one or more cryoprotectants that are the same or different.
• the concentration of the one or more cryoprotectants in the first and the second solution can be the same or different, and normally the total concentration of the cryoprotectant in the second solution is larger than that in the first solution.
• the cryoprotectant is trehalose.
• concentration of trehalose contained in the first and/or the second solution is normally within a range from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.2 M to about 0.7 M, from about 0.3 M to about 0.65 M, from about 0.4 M to about 0.6 M, from about 0.45 M to about 0.55 M.
• sucrose is used in similar applications.
• Trehalose is a unique, naturally occurring disaccharide and is found in hundreds of plants and animals.
• Trehalose is an important source of energy and has been shown to be a primary factor in stabilization of organisms during time of freezing. It has been shown that trehalose can depress the phase transition temperature of membranes so that they remain in the liquid-crystal state even when dry. Without being bound to any theory, it is hypothesized that this prevents membrane leakage during rehydration, thereby preserving cellular viability. With respect to proteins, trehalose has been shown to inhibit protein denaturation by exclusion of water from the protein surface when the cells are in the hydrated state.
• the cryoprotectant is sucrose.
• concentration of sucrose contained in the first and/or the second solution is normally within a range from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.2 M to about 0.7 M, from about 0.3 M to about 0.65 M, from about 0.4 M to about 0.6 M, from about 0.45 M to about 0.55 M.
• At least one of the first and second solutions comprises a cryoprotectant.
• At least one of the first and the second solution may comprise a viscosity-adjusting agent.
• Suitable viscosity-adjusting agent for use in the present context may be selected from the group consisting of Ficoll, Percoll, hyaluronic acid, albumin, polyvinyl pyrrolidone, alginic acid, gelatin and glycerol.
• the first and the second solution may contain one or more viscosity-adjusting agents that are the same or different.
• the concentration of the one or more viscosity-adjusting agents in the first and the second solution may be the same or different.
• the viscosity-adjusting agent is Ficoll.
• the concentration of Ficoll contained in the first and/or the second solution is at the most about 150 mg/ml, such as, e.g., at the most about 100 mg/ml, at the most about 50 mg/ml, at the most about 25 mg/ml, at the most about 15 mg/ml or at the most about 10 mg/ml.
• At least one of the first and second solutions is an aqueous solution.
• step ii) of the above-mentioned method is included.
• a possible time span would be 10 sec - 20 min since the point with this step is to promote equilibration with the solution and to ensure that the cryoprotectants sufficiently perfuses, but if DMSO is present the cells should not to be exposed to the somewhat toxic DMSO too long.
• the incubation is normally performed at about 37 °C for a time period from between 5 sec to about 20 min such as, e.g., from about 10 sec to about 15 min, from about 15 sec to about 10 min, from about 20 sec to about 7.5 min, from about 30 sec to about 5 min, from about 40 sec to about 4 min, from about 50 sec to about 3 min, from about 30 sec to about 2 min, from about 45 sec to about 1.5 min or about 1 min.
• 5 sec to about 20 min such as, e.g., from about 10 sec to about 15 min, from about 15 sec to about 10 min, from about 20 sec to about 7.5 min, from about 30 sec to about 5 min, from about 40 sec to about 4 min, from about 50 sec to about 3 min, from about 30 sec to about 2 min, from about 45 sec to about 1.5 min or about 1 min.
• step iv) is also included, and the incubation is normally performed at about 37 °C for a time period from between about 5 sec to about 10 min such as, e.g., from about 10 sec to about 7.5 min, from about 10 sec to about 5 min, from about 15 sec to about 4 min, from about 15 sec to about 3 min, from about 15 sec to about 2 min, from about 20 sec to about 1 min, from about 5 sec to about 1 min, from about 5 sec to about 30 sec or from about 10 sec to about 30 sec.
• 5 sec to about 10 min such as, e.g., from about 10 sec to about 7.5 min, from about 10 sec to about 5 min, from about 15 sec to about 4 min, from about 15 sec to about 3 min, from about 15 sec to about 2 min, from about 20 sec to about 1 min, from about 5 sec to about 1 min, from about 5 sec to about 30 sec or from about 10 sec to about 30 sec.
• step iv) is included and the incubation is performed at about 37 °C for about 30 sec or less.
• the vitrification method is very efficient and mild to the cells. Normally, about 50% or more such as, e.g., about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more or about 95% or more of the cells are viable after being devitrified and cultures in a suitable medium.
• a suitable devitrification procedure is described herein.
• the invention also relates to a cell, which has undergone vitrification by the method according to the invention.
• the devitrification comprises viii) subjecting one or more vitrified closed straw to an environment having a temperature of from about room temperature to about 40 °C for a time period of that allows the content of the closed straw to thaw, ix) opening of the one or more closed straw, x) subjecting the cells contained in the one or more opened closed straw to a washing procedure using a third solution (solution C), xi) optionally transferring the washed cells obtained from step x) to a fourth solution
• solution D (solution D), and xii) optionally incubating the cells in the fourth solution, xiii) optionally transferring the cells from xii) from the fourth solution and seeding the cells on feeder cells, and xiv) optionally further cultivating the cells.
• step x) The cells obtained after step x) are ready to use for whatever purpose that is desired and the optional step may be applied in order to investigate the cells further e.g. for viability.
• Step viii) concerns the thawing of the cells.
• the point here is just to thaw the content of the straw and this should be carried out during visual inspection of the straw, thus the timing is less important.
• the temperature should not be greater that 40 °C but can be between room temperature and 40 °C. Higher temperatures could induce a heat shock when thawing the cells if they are not rapidly removed from the water bath after thawing.
• the method may also - as mandatory steps - comprise steps xi), xiii) and xiv); and, furthermore, step xii).
• the devitrification solutions may contain cryoprotectants e.g. cryoprotectants having osmotic activity such as osmotically active agents with low toxicity, generally avoiding e.g. DMSO, and preferentially using e.g. trehalose and sucrose, alone or in combination.
• the high percentage of the disaccharides in this solution prevents cellular disruption that otherwise would occur by the sudden contact with a solution without DMSO.
• the presence of the disaccharides outside the cells will prevent the natural osmotic force from acting and will allow enough time for the cells to discard the DMSO (or similar) present inside the cells and substitute it slowly by water.
• the third and/or fourth (if relevant) solutions normally comprise one or more cryoprotectants.
• the one or more cryoprotectant is selected from the group consisting of glycerol, trehalose, sucrose, ethylene glycol, DMSO, propanediol, and or mixtures thereof, especially glycerol, trehalose, sucrose, or mixtures thereof is suitable for use.
• the concentration of the cryoprotectant in the third and/or fourth solution is normally from about 0.02 M to about 1 M such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.7 M, from about 0.1 M to about 0.6 M, from about 0.15 M to about 0.5 M, from about 0.2 M to about 0.4 M, and the concentration of the cryoprotectant in the third solution is larger than the concentration of the osmotically active agent in the fourth solution, if relevant.
• the concentration of the cryoprotectant in the third solution is larger than the concentration of the cryoprotectant in the fourth solution, if relevant.
• Colonies of human blastocyst-derived stem (hBS) cells are cut into pieces (0.1 -0.4mm x 0.1 -0.4mm). Up to 20 (preferably about 10) cell pieces in a volume of 40-50 ⁇ l can be frozen in a closed straw.
• a closed straw has a stopper in one end and is open in the other. After the cell pieces have been aspirated into the straw for freezing, the end with the plug (stopper) is sealed using cryo-PBS, while the open end is sealed using a bond (weld) and a Heatseal apparatus (Demtek, A/S). Before a larger amount of cells are frozen a test freezing and thawing round is performed. After thawing, the cell pieces are seeded onto a culture dish with mouse embryonic feeder cells (MEF). The human BS cells are cultured for one passage and are then evaluated.
• MEF mouse embryonic feeder cells
• a stock solution consisting of 0.6M Trehalose in Cryo-PBS obtained from Vitrolife AB, Gothenburg, Sweden is prepared.
• Solution A 10% Ethylene glycol is prepared in cryo-PBS and sterile filtered.
• Sterile DMSO is added to a final concentration of 10%
• Solution B A solution consisting of 0.3M Trehalose and 20% Ethylene glycol is prepared in cryo-PBS and sterile filtered. Sterile DMSO is added to a final concentration of 20%
• Selected colonies of human blastocyst-derived stem cells which display proper morphology, are cut in the same way as when the cells are cut for passage using an autoclaved drawn glass capillary (World Precision Instruments) (or a stem cell cutting tool from Swemed).
• the cutting tool from Swemed is a sterile sharpened glass capillary, with a 25 degree angle and a 200 or 300 micrometer lumen, designed for cutting, manipulation, and transfer of hBS colonies, or parts of hBS colonies. It is produced by Swemed Lab International AB, Billdal, Sweden.
• the cells that are to be frozen in a straw are transferred, using a glass capillary (World Precision Instruments) or a drawn glass pipette (Pasteur, VWR International), to Solution A.
• the cells are then transferred to one drop (25 ⁇ l) of Solution B, and within 25 s the cells are transferred to another fresh drop of solution B (25 ⁇ l).
• the cells are incubated in Solution B for 25 s (maximum).
• the preferred time for point 3-4 should be as short as possible.
• a 1-1.5 cm silicone tubing (autoclaved) is connected to a 1ml syringe (tuberculin- syringe, single-use, Codan Triplus AB), which in turn is connected to the straw in the end with the cotton stopper (plugged end).
• the silicone tubing serves as a seal between the straw and the syringe.
• cryo-PBS is aspirated into the straw in an approximately 2-3 cm column. Approximately, 1-2 cm air is then aspirated (see Figure 1).
• the cells are then aspirated into the straw from solution B under a stereomicroscope in a 2 cm column.
• cryo-PBS which serves as an extra stopper in that end.
• the content of the straw is aspirated with the syringe so that the cryo-PBS comes in contact with the cotton stopper, which makes the stopper swell.
• the straw is removed from the syringe using a pair of forceps and then sealed with a weld using a Heatseal apparatus.
• the straw is placed in a visiotube, which in turn is placed in a tank containing liquid nitrogen for long term storage.
• a stock solution consisting of 0.2M Trehalose in Cryo-PBS is prepared.
• Solution C 0.2M Trehalose in cryo-PBS is sterile filtered
• Solution D 0.1 M Trehalose in cryo-PBS is prepared and sterile filtered
• hBS medium contains KNOCKOUT ® Dulbecco's Modified Eagle's Medium, supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 100 units/ml penicillin, 0,1 mM non-essential amino acids, 2 mM L-glutamine, 100 ⁇ M ⁇ -mercaptoethanol, 4 ng/ml human recombinant bFGF (basic fibroblast growth factor).]
• the concentrations can also be varied (5-50% v/v, w/w or w/v) with different efficiencies. It would also be possible to use other cryoprotectants with low toxicity.
• the closed straw is held in the air at room temperature for 10 s and is then placed in a water bath at 40 ° C for about 2 sec.
• the straw is dried using an autoclaved "all purpose rag" (allduk).
• the hBS cell colonies are incubated in solution C for 1 min. Normally from about 1 min to about 20 min. 5. Under a stereo-microscope, the hBS cell colonies are transferred to solution D using a glass capillary (World Precision Instruments) or a drawn glass pipette (Pasteur, VWR International).
• the hBS cell colonies are incubated in solution D for 5 min. Normally from about 5 min to about 30 min.
• the hBS cell colonies are transferred to hBS-medium using a glass capillary (World Precision Instruments) or a drawn glass pipette (Pasteur, VWR).
• the colonies are within a few seconds (>5 s) transferred from the hBS-medium using a glass capillary (World Precision Instruments) or a drawn glass pipette (Pasteur, VWR International) and are seeded in a culture dish on top of mouse embryonic feeder cells (MEF).
• a glass capillary World Precision Instruments
• a drawn glass pipette Pasteur, VWR International
• the culture dish is placed in an incubator for further cultivation.
• Figure 1 Thawing recovery after vitrification and devitrification, human BS cell line SA001.
• Figure 2 Thawing recovery after vitrification and devitrification, human BS cell line SA002.
• Figure 3 Thawing recovery after vitrification and devitrification, human BS cell line AS034.
• Figure 6 Typical morphology of human BS cell line SA001 after devitrification cultured on mouse embryonic feeder cells in passage 18 (A), passage 23 (B), passage 29 (C), and passage 35 (D).
• Figure 7 (A) human BS cell colony [p19], (B) SSEA-1 [p31], (C) SSEA-3 [p31], (D) SSEA-4 [p31], (E) TRA-1-60 [p31], (F) TRA-1-81 [p31], (G) Oct-4 [p31], (H) ALP [p31]
• Figure 8 Karyotype, cell line SA001 after vitrification
• Figure 9 In vitro differentiation of devitrified human BS cells (SA001), passage 29.
• SA001 devitrified human BS cells
• A ⁇ - lll-tubulin
• B desmin
• C ⁇ -fetoprotein
• D HNF-3 ?
• FIG 10 In vivo differentiation of human BS cells (SA001), passage 19.
• A Endoderm (secretory epithelium),
• B Mesoderm (cartilage),
• C Ectoderm (neuroectoderm).
• FIG. 11 Syringe with a closed straw prepared for freezing
• the cell pieces are incubated first in 500 ⁇ l preheated (37°C) Solution A for 1 min and then transferred to 25 ⁇ l Solution B and incubated for 30 s and then transferred again to a fresh drop of Solution B and incubated for 30 s.
• the volume is about 40-50 ⁇ l.
• About 10 cell pieces are aspirated into a straw prepared for vitrification and the straw is then closed with a bond. The straw is plunged into liquid nitrogen.
• Solutions C and D are prepared (Solution C: Sterile filtered 0.2M Trehalose in Cryo- PBS.; Solution D: Sterile filtered 0.1M Trehalose in Cryo-PBS). Solutions C and D and hBS-medium are preheated at 37 °C. A closed straw containing vitrified hBS cells (about 10 cell pieces) is removed from the liquid nitrogen tank. The straw is keep at room temperature for 10 s and then quickly thawed in a 40 °C water bath (within seconds). The straw is cut open in the plugged end using an autoclaved pair of scissors and the content pushed out from the straw into solution C using a syringe.
• the hBS cells are incubated for 1 min in 500 ⁇ l solution C and the transferred to 500 ⁇ l solution D and incubated for 5 min. Under a stero-microscope the hBS cell pieces are quickly rinsed in hBS medium and then seeded in a culture dish on top of mouse embryonic feeder cells in hBS medium. The cells are then cultured (incubated at 37 °C) and the number of established new colonies are counted and passaged in order to verify the viability of the hBS cells after vitrification. The recovery of viable cells following this vitrifying and thawing procedures is normally in the range of 70-100%.
• Human BS cells (cell lines SA002, SA121 , and SA181) were vitrified and thawed following the procedure described in Example 1 & 2. Forty-eight hours after seeding in culture dishes on top of mouse embryonic feeder cells the hBS cell colonies were evaluated and counted. The thawing recovery was calculated as the ratio between the number of viable thawed colonies (displaying appropriate hBS cell morphology) and the number of hBS cell pieces originally vitrified, since each of these cell pieces can give rise to one colony. Three straws were prepared and evaluated per cell line and the results are presented below for each individual straw, showing a recovery of between 40% and 100%.
• Human BS cells (cell line AS034) were vitrified and thawed following the procedure described in Example 1 & 2 with the exception that open pulled straws were used in parallel to closed straws. Notably, only approximately 4 BS cell pieces can be vitrified in each open pulled straw. Forty-eight hours after seeding in culture dishes on top of mouse embryonic feeder cells the hBS cell colonies were evaluated and counted. The thawing recovery was calculated as the ratio between the number of viable thawed colonies (displaying appropriate hBS cell morphology) and the number of hBS cell pieces originally vitrified. Three straws were prepared and evaluated per cell line and the results are presented below for each individual straw and show the achievement of obtaining more viable cells from each of the closed straws (in absolute numbers) while maintaining an acceptable recovery.
• Human BS cells (cell line SA121) were vitrified and thawed either following the procedure described in Example 1 & 2 or following the procedure described in Example 1 and 2 with the exception that trehalose was used in the vitrification and devitrification medium was replaced by sucrose in the same molar concentration as used for trehalose.
• Three straws were prepared and evaluated per cell line and the results are presented below for each individual straw and shows that in this case there seems to be no significant difference between using trehalose and sucrose.
• Human BS cells (cell lines SA121) were vitrified and thawed following the procedure described in Example 1 & 2 with the exception that Ficoll was used in the vitrification medium (Omg/ml, 10mg/ml, and 10Omg/ml). Forty-eight hours after seeding in culture dishes on top of mouse embryonic feeder cells the hBS cell colonies were evaluated and counted. The thawing recovery was calculated as the ratio between the number of viable thawed colonies (displaying appropriate hBS cell morphology) and the number of hBS cell pieces originally vitrified. Three straws were prepared and evaluated per cell line and the results are presented below for each individual straw.
• Human BS cells (cell line SA121) were vitrified and thawed following the procedure described in Example 1 & 2 with the exception that trehalose was used in two different concentrations (0.3M and 0.5M) in the second vitrification solution (solution B).
• solution B When 0.3 M trehalose was used in solution B, solution C contained 0.2 M trehalose and solution D contained 0.1 M trehalose.
• solution C When 0.5 M trehalose was used in solution B, solution C contained 0.4 M trehalose and solution D contained 0.2 M trehalose. Forty-eight hours after seeding in culture dishes on top of mouse embryonic feeder cells the hBS cell colonies were evaluated and counted.
• the thawing recovery was calculated as the ratio between the number of viable thawed colonies (displaying appropriate hBS cell morphology) and the number of hBS cell pieces originally vitrified.
• Two separate experiments using two different human BS cell lines were performed. Three straws were prepared and evaluated per cell line and the results are presented below for each individual straw. The results show that 0.5 M trehalose in solution B seems to work better than 0.3 M trehalose in solution B, although both of the investigated trehalose conditions work well.
• Example 9 Typical morphology of human BS cell before and after vitrification and thawing
• Typical morphology of the human BS colonies (cell line SA001) before vitrification is shown in Figure 4. After devitrification and seeding, viable colonies proliferated and displayed morphology characteristic for undifferentiated human BS cells ( Figure 5). Subsequently, these cells were propagated and passaged according to standard procedures and representative illustrations of the human BS cell colonies are shown in Figure 6. Similar results were obtained for human BS cell line SA002 and AS034 (data not shown).
• the hBS cells In order to verify that the human BS cells completely recover and display the proper characteristics after the vitrification and devitrification process, the hBS cells subjected to extensive characterization. This includes analysis of surface antigen expression, karyotyping, and pluripotency tests in vitro as well as in vivo. The results below were obtained using human BS cell line SA001 , and similar results were also obtained using human BS cell lines SA002 and AS034 (data not shown).
• Devitrified human BS cells (cell line SA001) cultured on mouse embryonic feeder (MEF) cells were fixed in PFA and subsequently permeabilized using Triton X-100. After consecutive washing and blocking steps, the cells were incubated with the primary antibody (as indicated). Conjugated secondary antibodies were subsequently used for detection. The nuclei were visualized by DAPI staining.
• the activity of alkaline phosphatase (ALP) was determined using a commercial available kit following the instructions indicated by the manufacturer (Sigma Diagnostics, Sweden). The passage number at which each analysis was performed is indicated within brackets in the figure legend to Figure 7.
• the results show that the human BS cells displayed positive staining for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 , Oct-4, and ALP and that they were negative for SSEA-1 as expected for undifferentiated human BS cells.
• Devitrified human BS cells (line SA001) cultured on MEF were incubated in the presence of Calyculin A and then washed with cell culture medium. The cells were collected by centrifugation and fixed using ethanol and glacial acetic acid. The chromosomes were visualized using a trypsin-Giemsa staining. As illustrated in Figure 8, the results show that there were no detectable chromosomal abnormalities in the cells following the vitrification and devitrification process.
• telomerase activity For analyzing the telomerase activity a Telo TAGGG Telomerase PCR ELISA PLUS kit (Roche, Basel, Switzerland) was employed according to the manufacturer's instructions. The assay uses the internal activity of telomerase, amplifying the product by PCR and detecting it with an enzyme linked immunosorbent assay (ELISA). Human BS cell line SA001 was analyzed after devitrification and culture on mouse embryonic feeder cells and displayed high telomerase activity. High telomerase activity in hBS cells correlates with their ability to divide indefinitely in culture.
• ELISA enzyme linked immunosorbent assay
• the method for establishing pluripotent human blastocyst-derived stem cells or cell line from a fertilized oocyte comprises the steps of i) using a fertilized oocyte optionally, having a grade 1 or 2, to obtain a blastocyst, optionally having a grade A or B, ii) co-culturing the blastocyst with feeder cells for establishing one or more colonies of inner cell mass cells, iii) isolating the inner cell mass cells by mechanical dissection, iv) co-culturing of the inner cell mass cells with feeder cells to obtain a blastocyst- derived stem cell line, v) optionally, propagation of the blastocyst-derived stem cell line.
• fertilized oocytes are used.
• the quality of the fertilized oocytes is of importance for the quality of the resulting blastocysts.
• the human blastocysts in step i) of the method may be derived from frozen or fresh human in vitro fertilized oocytes.
• an important success criterion for the present method is a proper selection of oocytes.
• the probability of obtaining a hBS cell line fulfilling the general requirements (described below) is low.
• Donated fresh fertilized oocytes On day 0 the oocyte is aspirated in Asp-100 (Vitrolife), and fertilized on day 1 in IVF-50 (Vitrolife). The fertilized oocyte is evaluated based on morphology and cell division on day 3. The following scale is used for fertilized oocyte evaluation:
• Grade 1 fertilized oocyte Even blastomers, no fragments Grade 2 fertilized oocyte: ⁇ 20% fragments Grade 3 fertilized oocyte: >20% fragments
• fertilized oocytes of grade 1 and 2 are either implanted or frozen for storage. Fertilized oocytes of grade 3 are transferred to ICM-2 (Vitrolife). The fertilized oocytes are further cultured for 3-5 days (i.e. day 5-7 after fertilization). The blastocysts are evaluated according to the following scale:
• Donated frozen fertilized oocytes At day 2 (after fertilization) the fertilized oocytes are frozen at the 4-cell stadium using Freeze-Kit (Vitrolife). Frozen fertilized oocytes are stored in liquid nitrogen. Informed consent is obtained from the donors before the 5-year limit has passed. The fertilized oocytes are thawed using Thaw-Kit (Vitrolife), and the procedure described above is followed from day 2.
• Freeze-Kit Vitrolife
• fresh fertilized oocytes are from grade 3 quality
• frozen fertilized oocytes are from grade 1 and 2.
• the percentage of fresh fertilized oocytes that develop into blastocysts is 19%, while 50% of the frozed fertilized oocytes develop into blastocysts.
• the frozen fertilized oocytes are much better for obtaining blastocysts, probably due to the higher quality of the fertilized oocytes.
• 11 % of the blastocysts derived from fresh fertilized oocytes develop into a stem cell line
• 15% of the blastocysts derived from frozen fertilized oocytes develop into a stem cell line.
• the fertilized oocytes that were put into culture 2% of fresh fertilized oocytes developed into a stem cell line, and 7% of frozen fertilized oocytes that were put into culture developed into a stem cell line.
• the culturing of the fertilized oocyte to the blastocyst-stage is performed after procedures well-known in the art. Procedures for preparing blastocysts may be found in Gardner et al, Embryo culture systems, In Trounson, A. O., and Gardner, D. K. (edsj, Handbook of in vitro fertilization, second edition. CRC Press, Boca Raton, pp.
• the blastocysts having grade A or B are co-cultured with feeder cells for establishing one or more colonies of inner cell mass cells.
• feeder cells After being plated onto feeder cells, their growth is monitored and when the colony is large enough for manual passaging (approximately 1-2 weeks after plating), the cells may be dissected from other cell types and expanded by growth on new feeder cells.
• the isolation of the inner cell mass cells is performed by mechanical dissection, which may be performed by using glass capillaries as a cutting tool.
• the detection of the inner cell mass cells is easily performed visually by microscopy and, according, it is not necessary to use any treatment of the oocytes with enzymes and/or antibodies to impair or remove the trophectoderm.
• the inner cell mass cells are co-cultured with feeder cells to obtain a blastocyst-derived stem (BS) cell line.
• BS blastocyst-derived stem
• the cell line is optionally propagated to expand the amount of cells.
• the blastocyst-derived stem cell line may be propagated e.g. by passage of the stem cell line every 4-5 days. If the stem cell line is cultured longer than 4-5 days before passage, there is an increased probabilily that the cells undesirably will differentiate.
• a specific procedure of passaging the cells in a feeder culture system is given in Establishment example 5 herein.
• Human BS cell lines may be isolated either from spontaneously hatched blastocysts or from expanded blastocysts with an intact zona pellucida.
• the blastocyst in step i) is a spontaneously hatched blastocyst.
• the trophectoderm may be left intact.
• Either hatched blastocysts or blastocysts with a removed or partially removed zona pellucida may be put on inactivated feeder cells.
• Zona pellucida of the blastocyst may be at least partially digested or chemically frilled prior to step ii) e.g. by treatment with one or more acidic agents such as, e.g., ZDTM-10 (Vitrolife, Gothenburg, Sweden), one or more enzymes or mixture of enzymes such as pronase.
• one or more acidic agents such as, e.g., ZDTM-10 (Vitrolife, Gothenburg, Sweden)
• one or more enzymes or mixture of enzymes such as pronase.
• Other types of proteases with the same or similar protease activity as pronase may also be used.
• the blastocysts can be plated onto said inactivated feeder cells following the pronase treatment.
• step ii) and/or step iv) may be performed in an agent that improves the attachment of the blastocysts and/or if relevant the inner cell mass cells to the feeder cells.
• a suitable substance for this purpose is a hyaluronic acid.
• a suitable medium for plating the blastocysts onto feeder cells can be hBS-medium that may be complemented with hyaluronic acid, which seems to promote the attachment of the blastocysts on the feeder cells and growth of the inner cell mass.
• Hyaluronan (HA) is an important glycosaminoglycan constituent of the extracellular matrix in joints. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM), and to proteins in the extracellular matrix.
• HA may be exerted through its interactions with the surfactant polar heads of phospho ⁇ pids in the cell membrane, to thereby stabilize the surfactant layer and thus lower the surface tension of the inner cell mass or blastocyst which may result in increased efficiency in binding to the feeder cells.
• HA may bind to its receptors on the inner cell mass or blastocyst and/or to the feeder cells and exert biological effects which positively influence the attachment and growth of the inner cell mass.
• other agents that may alter the surface tension of fluids, or in other ways influence the interaction between the blastocyst and feeder cells can also be used in instead of hyaluronic acid.
• the propagation of blastocyst-derived stem cell line may comprise passage of the feeder cells at the most 3 times, such as e.g. at the most 2 times.
• Suitable feeder cells for use in a method of the invention are fibroblasts of e. g. embryonic or adult origin.
• the feeder cells employed in steps ii) and iv) are the same or different and originate from animal source such as e.g. any mammal including human, mouse, rat, monkey, hamster, frog, rabbit etc. Feeder cells from human or mouse species are preferred.
• the blastocyst-derived stem cell line may accordingly by propagated by culturing the stem cells with feeder cells of a density of less than about 60,000 cells per cm 2 , such as e.g. less than about 55,000 cells per cm 2 , or less than about 50,000 cells per cm 2 .
• the propagation of blastocyst-derived stem cell line comprises culturing the stem cells with feeder cells of a density of about 45,000 cells per cm 2 .
• the blastocyst-derived stem cell line obtained by the establishment method described above maintains selfrenewal and pluripotency for a suitable period of time and, accordingly it is stable for a suitable period of time.
• stable is intended to denote proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells.
• the stem cell line obtained by the establishment method described above fulfils the general requirements.
• the cell line i) exhibits proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells, and ii) exhibits normal euploid chromosomal karyotype, and iii) maintains potential to develop into derivatives of all types of germ layers both in vitro and in vivo, and iv) exhibits at least two of the following molecular markers OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 , and the protein core of a keratin sulfate/chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody GCTM-2, and v) does not exhibit molecular marker SSEA-1 or other differentiation markers, and vi) retains its pluripotency and forms teratomas in vivo when injected
• the undifferentiated hBS cells obtained by the method described above are defined by the following criteria; they were isolated from human pre-implantation fertilized oocytes, i.e. blastocysts, and exhibit a proliferation capacity in an undifferentiated state when grown on mitotically inactivated feeder cells; they exhibit a normal chromosomal karyotype; they express typical markers for undifferentiated hBS cells, e.g.
• OCT-4 alkaline phosphatase
• carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 the carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 , and the protein core of a keratin sulfate/chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody GCTM-2, and do not show any expression of the carbohydrate epitope SSEA-1 or other differentiation markers.
• pluripotency tests in vitro and in vivo demonstrate differentiation into derivatives of all germ layers.
• the method proveds an essentially pure preparation of pluripotent human BS cells, which i) exhibits proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells; ii) exhibits normal euploid chromosomal karyotype; iii) maintains potential to develop into derivatives of all types of germ layers both in vitro and in vivo; iv) exhibits at least two of the following molecular markers OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 , and the protein core of a keratin sulfate/chondroitin sulfate pericellular matrix proteinglycan recognized by the monoclonal antibody GCTM-2 v) does not exhibit molecular marker SSEA-1 or other differentiation markers, and vi) retains its pluripotency and forms teratomas in vivo when injected into immuno-
• Procedures for the detection of cell markers can be found in Gage, F. H., Science, 287:1433-1438 (2000). These procedures are well known for the skilled person and include methods such as RT-PCR or immunological assays where antibodies directed against the cell markers are used. In the following, methods for detection of cell markers, hybridisation methods, karyotyping, methods for measuring telomerase activity and teratoma formation are described. These methods can be used to investigate whether the hBS cells obtained according to the establishment method fulfil the above-mentioned criteria.
• the hBS stem cells maintained in culture are routinely monitored regarding their state of differentiation.
• Cell surface markers used for monitoring the undifferentiated hBS cells are SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81.
• Human BS stem cells are fixed in 4% PFA and subsequently permeabilized using 0.5% Triton X-100. After washing and blocking with 10% dry milk the cells are incubated with the primary antibody. After extensive washes the cell are incubated with the secondary antibody and the nuclei are visualized by DAPI staining.
• alkaline phosphatase The activity of alkaline phosphatase is determined using a commercial available kit following the instructions from the manufacturer (Sigma Diagnostics).
• mRNA levels for the transcription factor Oct-4 is measured using RT-PCR and gene specific primer sets (5'-CGTGAAGCTGGAGAAGGAGAAGCTG,
• FISH Fluorescence In situ Hybridization
• chromosome specific probes In one round of FISH one ore more chromosomes are being selected with chromosome specific probes. This technique allows numerical genetic aberrations to be detected, if present.
• CTS uses a commercially available kit containing probes for chromosome 13, 18, 21 and the sex chromosomes (X and Y) (Vysis. Inc, Downers Grove, IL, USA). For each cell line at least 200 nuclei are being analyzed. The cells are resuspended in Camoy ' s fixative and dropped on positively charged glass slides. Probe LS1 13/21 is mix with LSI hybridization buffer and added to the slide and covered with a cover slip.
• Probe CEP X/Y/18 is mixed with CEP hybridization buffer and added in the same way to another slide. Denaturing is performed at 70°C for 5 min followed by hybridization at 37°C in a moist chamber for 14-20h. Following a three step washing procedure the nuclei are stained with DAPI II and the slides analyzed in an invert microscope equipped with appropriate filters and software (CytoVision, Applied Imaging).
• Karyotyping allows all chromosomes to be studied in a direct way and is very informative, both numerical and larger structural aberrations can be detected. In order to detect mosaicism, at least 30 karyotypes are needed. However, this technique is both very time consuming and technically intricate.
• the mitotic index can be raised by colcemid, a synthetic analog to colchicin and a microtubule- destabilizing agent causing the cell to arrest in metaphase, but still a large supply of cells are needed (6x10 6 cells/analysis).
• the cells are incubated in the presence of 0.1 ⁇ g/ml colcemid for 1-2h, and then washed with PBS and trypsinized. The cells are collected by centrifugation at 1500rpm for 10min. The cells are fixed using ethanol and glacial acetic acid and the chromosomes are visualized by using a modified Wrights staining.
• Comparative genomic hybridization is complementary to karyotyping. CGH gives a higher resolution of the chromosomes and is technically less challenging. Isolated DNA is nicktranslated in a mixture of DNA, A4, Texas red -dUTP/ FITC 12-dUTP, and DNA polymerase I. An agarose gel electrophoresis is performed to control the size of resulting DNA fragments (600-2000 bp). Test and reference DNA is precipitated and resuspended in hybridization mixture containing formamide, dextrane sulfate and SSC. Hybridization is performed on denatured glass slides with metaphases for 3 days at 37°C in a moist chamber. After extensive washing one drop of antifade mounting mixture (vectashield, 0,1 ⁇ g/ml DAPI II) is added and the slides covered with cover slips. Slides are subsequently evaluated under a microscope and using an image analysis system.
• Isolated DNA is nicktranslated in a mixture of DNA, A4, Texas red
• telomerase activity is measured in the hBS cell lines. It is known that telomerase activity successively decrease when the cell reaches a more differentiated state. Quantifying the activity must therefore be related to earlier passages and control samples, and can be used as a tool for detecting differentiation.
• the method Telomerase PCR ELISA kit (Roche) uses the internal activity of telomerase, amplifying the product by polymerase chain reaction (PCR) and detecting it with an enzyme linked immunosorbent assay (ELISA). The assay is performed according to the manufacturer's instructions. The results from this assay shows typically a high telomerase activity (>1) for hBS cells.
• the cell lines retain their pluripotency and forms teratomas in vivo when injected into immuno-compromised mice. In addition, in vitro these cells can form hBS cell derived bodies. In both of these models, cells characteristic for all germ layers can be found.
• One method to analyze if a human BS cell line has remained pluripotent is to xenograft the cells to immunodeficient mice in order to obtain tumors, teratomas.
• Various types of tissues found in the tumor should represent all three germlayers. Reports have showed various tissues in tumors derived from xenografted immunodeficient mice, such as striated muscle, cartilage and bone (mesoderm) gut (endoderm), and neural rosettes (ectoderm). Also, large portions of the tumors consist of disorganized tissue.
• Severe combined immunodeficient (SCID) -mice a strain that lack B- and T-lymphocytes are used for analysis of teratoma formation.
• Human BS cells are surgically placed in either testis or under the kidney capsule. In testis or kidney, hBS cells are transplanted in the range of 10 000-100 000 cells. Ideally, 5-6 mice are used for each cell line at a time. Preliminary results show that female mice are more post-operative stable than male mice and that xenografting into kidney is as effective in generating tumors as in testis. Thus, a female SCID-mouse teratoma model is preferable. Tumors are usually palpable after approximate 1 month.
• mice are sacrificed after 1-4 months and tumors are dissected and fixed for either paraffin-or freeze-sectioning.
• the tumor tissue is subsequently analyzed by immunohistochemical methods.
• Specific markers for all three germlayers are used.
• the markers currently used are: human E-Cadherin for distinction between mouse tissue and human tumour tissue, ⁇ -smooth muscle actin (mesoderm), ⁇ -Fetoprotein (endoderm), and ?-lll-Tubulin (ectoderm). Additionally, hematoxylin-eosin staining is performed for general morphology.
• BS-cell medium or "BS-medium” and may be comprised of; KNOCKOUT ® Dulbecco's Modified Eagle's Medium, supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids, 2 mM L-glutamine, 100 ⁇ M ⁇ -mercaptoethanol, 4 ng/ml human recombinant bFGF (basic fibroblast growth factor).
• KNOCKOUT ® Dulbecco's Modified Eagle's Medium supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids, 2 mM L-glutamine, 100 ⁇ M ⁇ -mercaptoethanol, 4 ng/ml human re
• BS cell body medium is "BS cell body medium”, this may be comprised as follows; KNOCKOUT ® Dulbecco's Modified Eagle's Medium, supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids, 2 mM L-glutamine and 100 ⁇ M ⁇ -mercaptoethanol.
• KNOCKOUT ® Dulbecco's Modified Eagle's Medium supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids, 2 mM L-glutamine and 100 ⁇ M ⁇ -mercaptoethanol.
• stable is intended to denote proliferation capacity in an undifferentiated state for more than 21 months when grown on mitotically inactivated embryonic feeder cells.
• Human blastocysts were derived from frozen or fresh human in vitro fertilized embryos. Spontaneously hatched blastocysts were put directly on feeder cells (EF) in hBS cell medium (KNOCKOUT Dulbecco's Modified Eagle's Medium, supplemented with 20% KNOCKOUT Serum replacement, and the following constituents at the final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0.1 mM non-essential amino acids, 2mM L-glutamine, 100 ⁇ M ⁇ -mercaptoethanol, 4ng/ml human recombinant bFGF (basic fibroblast growth factor), supplemented with 0.125 mg/ml hyaluronic acid. After plating the blastocysts on the EF cells, growth was monitored and when the colony was large enough for manual passaging approximately 1-2 weeks after plating) the inner cell mass cells were dissected from other cell types and expanded by growth on new EF cells
• Establishment example 2 Establishment of an essentially pure preparation of undifferentiated stem cells from blastocysts with an intact zona pellucida
• a brief pronase (10 U/ml, Sigma) incubation in rS2 (ICM-2) medium (Vitrolife, Gothenburg, Sweden) was used to digest the zona, after which the blastocyst was put directly on the EF cell layer in hBS medium supplemented with hyaluronic acid (0.125 mg/ml).
• RNA isolation and RT-PCR Total cellular RNA was prepared using Rneasy Mini Kit (Qiagen) according to the manufacturer's recommendations.
• the cDNA synthesis was carried out using AMV First Strand cDNA Synthesis Kit for RT-PCR (Roche) and PCR using Platinum Taq DNA Polymerase (lnvitrogen). Histochemical staining for alkaline phosphatase was carried out using commercially available kit (Sigma) following the manufacturer's recommendations.
• Mouse embryonic fibroblasts feeder cells were cultivated on tissue culture dishes in EMFI- medium: DMEM (Dulbecco's Modified Eagle's Medium), supplemented with 10% FCS (Fetal Calf Serum), 0,1 ⁇ M ⁇ -mercaptoehanol, 50 units/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-glutamine (GibcoBRL).
• the feeder cells were mitotically inactivated with Mitomycin C (10 ⁇ g/ml, 3 hrs). Human BS cell-colonies were expanded by manual dissection onto inactivated mouse embryonic fibroblasts feeder cells.
• Human BS cells were cultured on mitotically inactivated mouse embryonic fibroblasts feeder cells in tissue culture dishes with hBS-cell medium: KNOCKOUT ® Dulbecco's Modified Eagle's Medium, supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids, 2mM L-glutamine, 100 ⁇ M ⁇ - mercaptoethanol, 4 ng/ml human recombinant bFGF (basic fibroblast growth factor). Seven days after passage the colonies were large enough to generate BS cell bodies.
• KNOCKOUT ® Dulbecco's Modified Eagle's Medium supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids
• BS cell colonies were cut with glass capillaries into 0.4x0.4 mm pieces and plated on non- adherent bacterial culture dishes containing BS cell body medium: KNOCKOUT ® Dulbecco's Modified Eagle's Medium, supplemented with 20% KNOCKOUT ® Serum replacement and the following constituents at their respective final concentrations: 50 units/ml penicillin, 50 ⁇ g/ml streptomycin, 0,1 mM non-essential amino acids, 2 mM L- glutamine and 100 ⁇ M ⁇ -mercaptoethanol.
• the BS cell bodies including cystic hBS cell bodies, formed over a 7-9-day period.
• the hBS cells Before passage the hBS cells are photographed using a Nikon Eclipse TE2000-U inverted microscope (10X objective) and a DXM 1200 digital camera. Colonies are passaged every 4-5 days. The colonies are big enough to be passaged when they can be cut in pieces (0.1-0.3 x 0.1-0.3 mm). The first time the cells are passaged, they have grown for 1-2 weeks and can be cut in approximately four pieces.
• the colonies are focused, one by one, in a stereo-microscope and cut in a checkered pattern according to the size above. Only the inner homogeneous structure is passaged. Each square of the colony is removed with the knife, aspirated into a capillary and placed on new feeder cells (with the maximum age of 4 days). 10-16 squares are placed evenly in every new IVF-dish. The dishes are left five to ten minutes so the cells can adhere to the new feeder and then placed in an incubator. The hBS medium is changed three times a week. If the colonies are passaged, medium is changed twice that particular week. Normally a "half change" is made, which means that only half the medium is aspirated and replaced with the equal amount of fresh, tempered medium. If necessary the entire volume of medium can be changed.
• EMFi embryonic mouse feeder
• the cells are inactivated with EMFi medium containing Mitomycin C by incubation at 37°C for 3 hours. IVF-dishes are coated with gelatin. The medium is aspirated and the cells washed with PBS. PBS is replaced with trypsin to detach the cells. After incubation, the trypsin activity is stopped with EMFi medium. The cells are then collected by centrifugation, diluted 1 :5 in EMFi medium, and counted in a B ⁇ rker chamber. The cells are diluted to a final concentration of 170K cells/ml EMFi medium. The gelatin in the IVF- dishes is replaced with 1 ml cell suspension and placed in an incubator. EMFi medium is changed the day after the seeding.
• hBS cells employed in the present invention may be cultured in a feeder-free culture system, which method is advantageous compared to the known methods in that the cells transferred are stable for at least up to 10 passages.
• Studies by Richards et al. showed that the hBS cell lines could not be propagated in an undifferentiated state for more than six passages on cell-free matrixes, including MatrigelTM.
• the hBS cells were stable for up to 35 passages on MatrigelTM, still expressing the markers for undifferentiated hBS cells, even after a cycle of freeze/thawing and growth rates remained roughly comparable. Furthermore, a significantly higher number of surviving colonies were observed two days after plating, when mechanical dissociation was compared with enzymatic dissociation. A critical step seems to bee the initial step for transfer of the hBS cells to a feeder-free culture system. Accordingly, below is described a method for transfer of hBS cells to a feeder-free culture system, wherein the hBS cells are mechanically cut from the feeder.
• Integrin alpha 1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices.
• Laminin-specific receptors possibly formed by Integrin ⁇ 6 and ?1 which are highly expressed by hBS cells, may also play a major role in the adhesion of hBS cell to the matrix surface.
• one possibility is that some of the important surface receptors for attachment or survival might be negatively affected by the rough initial Collagenase IV treatment before the cells have adapted to the new surface.
• different techniques for the transfer of hBS cells to a feeder-free environment were investigated, either by mechanical or enzymatical dissociation, in regards to cell adhesion, survival rate and proliferation.
• the method was developed in order to facilitate long-term propagation and large-scale production of homogenous populations of undifferentiated hBS cells. The use of conventional cryopreservation techniques for freezing/thawing of the hBS cells was also examined.
• the inner cell mass cells are co-cultured with feeder cells to obtain a blastocyst-derived stem (BS) cell line.
• BS blastocyst-derived stem
• the cell line is optionally propagated to expand the amount of cells.
• the hBS cells Before propagation of the hBS cells in a feeder-free system, the hBS cells may be transferred to a feeder-free system.
• a critical factor for the success in the propagation of the hBS cells is the method by which the hBS cells is transferred from a feeder culture system to a feeder-free culture system. Accordingly, the hBS cells must be transferred to the feeder-free culture system by mechanical dissection, which may be performed by using glass capillaries as a cutting tool. As shown in the examples herein, mechanical dissociation resulted in a much more efficient attachment of cells to the MatrigelTM, a more rapid proliferation compared to the enzyme treated cultures, and the cells were much more stable during passages. Accordingly, the method for transferring the HS cells according to the invention does not require any enzymatic treatment. As seen in the examples herein, the cells cultured and proliferated under feeder-free conditions have a mitotic index that was similar to that of cells grown under feeder conditions.
• the propagation of the blastocyst-derived stem cell line comprises culturing the stem cells under feeder cell free growth conditions, as culturing the hBS cells without feeder cells has a number of advantages, such as, e.g. there is no need for the ongoing production of feeder cells, the production of hBS cells may be easier to scale up to commercial production and there is no risk of DNA transfer or other infection risks from the feeder cells.
• the transfer and propagation step under feeder free conditions may comprise the following steps of a) transferring the blastocyst derived stem cells from feeder to feeder free culture by mechanical treatment. b) optionally, culturing the blastocyst derived stem cells under feeder cell free growth conditions in a suitable growth medium and/or on a suitable support substrate, and c) optionally, passaging the blastocyst derived stem cell line every 3-10 days by enzymatic and/or mechanical treatment.
• the transfer step has been found to be a critical step as mentioned above. Accordingly, the transfer should be done by means of mechanically dissociation or mechanical dissection of the cells in the feeder culture system.
• This mechanical treatment may be done by means of any suitable cutting tool such as a tool having a sharpened end and a size that is appropriate for the cutting.
• the tool may be made of any suitable material such as, e.g., plastic or glass and an example of a suitable tool is a cutting tool that is a sterile sharpened glass capillary, with a 25 degree angle and a 200 or 300 micrometer lumen, designed for cutting, manipulation, and transfer of hBS colonies, or parts of hBS colonies. It is produced by Swemed Lab International AB, Billdal, Sweden.
• the hBS cells to be transferred is a colony of hBS cells and pieces is cut from the centre of the colony and suspended in a suitable medium as cell clusters.
• the cell clusters are dissociated mechanically one or more times e.g. until the cell clusters have a size that is at least 50% such as, e.g., at the most about 40%, at the most about 30%, at the most about 20%, at the most about 10% or at the most about 5% of that of the orginical colony.
• the size is e.g. determined as the diameter of the cluster or colony, respectively.
• mEF cells for conditioning of VitroHESTM-medium
• a confluent monolayer of mEF cells (passage two) was Mitomycin C treated and seeded in a concentration of 59 000 cells/cm 2 in a gelatin (0.1%; Sigma) coated culture flask in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 1 % Penicillin/Streptomycin (PEST; lOOOOU/ml), 10% Fetal Bovine Serum (FBS) and 2 mM GLUTAMAX TM-l Supplement (200 mM); all from GibcoBRL/lnvitrogen, Carlsbad, CA, USA.
• D-MEM Dulbecco's Modified Eagle Medium
• FBS Fetal Bovine Serum
• 2 mM GLUTAMAX TM-l Supplement 200 mM
• VitroHESTM-medium (0.28 ml/ cm 2 ) for a 24 hour conditioning period.
• the conditioned VitroHESTM-medium (k-VitroHESTM-medium) was collected every day up to three times from the same mEF culture (in passage two) and sterile filtered by using a 0.2 ⁇ m low protein binding filter (Sarstedt, Landskrona, Sweden).
• the k-VitroHESTM-medium was used either fresh or after freezing at -20°C and supplemented with 4 ng/ml of bFGF (GibcoRL/lnvitrogen) prior to use.
• the k- VitroHESTM-medium may be used for up to one week if stored at +4°C. When stored at -20°C for up to two months, no sign of reduced bioreactivity could be detected upon usage.
• hBS cell lines were maintained on Mitomycin C treated mouse feeders in 10-50 passages and cultured in VitroHESTM-medium supplemented with 4ng/ml of human basic fibroblast growth factor (bFGF).
• bFGF human basic fibroblast growth factor
• the hBS cells were cut in square pieces, which represented the middle of the colony, by using a stem cell cutting tool (Swemed Lab AB, Billdal, Sweden), and carefully detached and transferred the cells to HBSS solution.
• the stem cell tool is a sterile sharpened glass capillary, with a 25 degree angle and a 200 or 300 micrometer lumen, designed for cutting, manipulation, and transfer of hBS colonies, or parts of hBS colonies. It is produced by Swemed Lab International AB, Billdal, Sweden.
• the cell clusters were carefully dissociated mechanically by using a 1-ml automatic pipette.
• the dissociation process was completed when the size of the cell clusters represented approximately 1/10 -1/20 of the original colonies (average of 20 000 cells/original colony) corresponding to the size of cell aggregates generated by Collagenase IV treatment, as described above
• the colonies were transferred to collagenase IV solution (200 U/ml) to start the enzyme dissociation.
• the cells were seeded into four wells each and incubated at 37°C in 5% CO 2 . Each experiment was repeated four times, with the same amount of cells seeded each time. After two and six days the colony size and number was calculated.
• the cells were then washed, resuspended in k-VitroHESTM medium and seeded at a split ratio of 1 :2 to 1 :6 onto MatrigelTM.
• the hBS cultures were passaged every 5 to 6 days and the medium was changed every second to third day.
• k-VitroHESTM-medium has to be prepared and preheated before thawing the cells by placing the cryotubes in 37° water bath until all of the cell suspension was thawed. The cell suspension was transferred to the preheated medium for 5 minutes before centrifugation (400 G in 5 minutes). MatrigelTM thin layer coated (BD) wells were rehydrated by adding 1 ml of k-VitroHESTM-medium to the wells and incubate 30 minutes in 37° C. The cell pellet was resuspended in k-VitroHESTM-medium and transferred to either 24- or 6-well MatrigelTM plates.
• BD thin layer coated
• Immunocytochemlstry The cultures were passaged as described above, seeded into 6- or 24-well MatrigelTM plates and cultured for six days before performing the immunostaining. The cultures were washed in PBS, fixed with 4% formaldehyde (HistoLab, Gothenburg, Sweden) for 15 minutes at room temperature and then washed again three times in PBS.
• the monoclonal primary antibodies used were directed against SSEA-1 , -3 and -4 (1 :200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), Tra-1-60, Tra- 1-81 (1 :200; Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal rabbit anti- Phospho-Histone H3 (1 :150; KeLab, Upstate).
• the primary antibodies were incubated over night at 4°C before being visualized using appropriate Cy3- or FITC- conjugated secondary antibodies (1 :300; Jackson ImmunoResearch Laboratories, West Grove, PA). Cultures were also incubated with 4'-6 ' Diamidino-2-phenylindole (DAPI; Sigma-Aldrich Sweden AB, Sweden), at a final concentration of 0.5 ug/mL for 5 minutes at room temperature, to visualize all the cell nuclei. The stained cultures were rinsed and mounted using DAKO fluorescent mounting medium (Dakopatts AB, Alvsj ⁇ , Sweden) and visualized in an inverted fluorescent microscope (Nikon Eclipse TE2000-U). Alkaline phosphatase (AP) staining of the MatrigelTM cultured hBS cells was carried out according to the manufacturer's instructions using a commercially available kit (Sigma-Aldrich).
• DAPI Diamidino-2-phenylindole
• telomerase activity MatrigelTM cultured hBS cells were harvested, lysed and telomerase activity analyzed by a PCR-based ELISA (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturers instructions.
• FISH fluorescence in situ hybridization
• Teratomas For the teratoma formation experiment, immunodeficient SCID mice (C.B- 17/lcrCrl-scidBR, Charles River Laboratories, Germany) were used. MatrigelTM propagated hBS colonies were enzymatically detached from the surface by using Collagenase IV (200U/ml), mechanically dissociated into small cell aggregates and approximately 50 000 to 100 000 cells/organ were injected under the kidney capsule. Control animals were treated with Cryo-PBS injections or with primary brain cells from a littermate. The animals were sacrificed eight weeks after injection and the tumors were immediately fixed in a 4 % solution of paraformaldehyde and paraffin embedded. For histological analysis the teratoma were sectioned to 8 ⁇ m and stained with Alcian Blue ⁇ /an Giesson.
• the PCR reaction included four initial step-down cycles, with two repeated cycles for every annealing temperature, with denaturation for 15 seconds at 94°C, annealing temperature for 15 seconds at 66° to 60°C and extension for 30 seconds at 72°C.
• the forward and reverse primer sequences for Oct-4 were previously described, ⁇ -actin primers were used as internal controls (sense, 5'-TGGCACCACACCTTCTACAATGAGC- 3'; antisense, 5'-GCACAGCTTCTCCTTAATGTC-ACGC-3'; 400 bp product).
• the PCR products were size fractioned by gel electrophoresis using a 1.5% agarose gel. Human liver was used as a positive control and water as negative control for the PCR reaction.
• Immunocytochemlstry SSEA-1 expression was negative in all feeder-free cultured hBS cell lines as opposed to staining with antibodies against SSEA-3, SSEA-4, TRA-1-60 and TRA 1-80 which show a clear positive immunoreaction as expected for pluripotent hBS cells. Further, the cells displayed high levels of AP reactivity in all four MatrigelTM propagated cell lines. Telomerase activity: Analysis was preformed on three of the MatrigelTM cultured hBS cell lines (AS 038, SA 121 and SA 167). The hBS cells cultured on MatrigelTM were found to have high levels of telomerase activity.
• Karyotyping and FISH Karyotype analysis was preformed on two of the MatrigelTM cultured cell lines, AS 038 and SA 121. Three of three cells from cell line AS 038 and ten of twelve cells from cell line SA 121 were found to possess normal human 46, XY karyotype (fig. 10). The remaining two cells from the SA 121 cell line expressed an abnormal karyotype of 45, XY and 42, XY. Although, karyotypic changes seem to be normal occurring events after prolonged culturing for both feeder and feeder-free hBS cell cultures.
• Teratoma formation was performed for two MatrigelTM cultured hBS cell lines, SA 167 and SA 002, and the results showed that teratomas formed consisting of differentiated cells and tissue representative from all three germ layers (endoderm, mesoderm and ectoderm, providing evidence that the MatrigelTM propagated hBS cultures have retained their pluripotency.
• Oct-4 expression was high in all four cell lines cultured on Matrigel TM
• Cell line SA 121 was cultured in parallel under feeder-free conditions on MatrigelTM coated plates and on embryonic mouse feeder cells for 3 days. The number of cells in mitosis was then quantified by nuclear immunoreactivity for phosphorylated Histone H3. The mitotic index in both cultures was calculated in order to compare the growth rate between feeder-free and feeder cultured hBS cells,

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