Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N5/0603—Embryonic cells ; Embryoid bodies
  • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• • 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

Definitions

• the present invention relates to placental stem cells and, more specifically, to a method for the procurement of placental stem cells.
• Stem cells are master cells found in all multicellular organisms. These special cells are important to the human body, for example, because they are capable of: (i) differentiating into a multitude of different specialized cell types; and (ii) dividing to maintain a supply of stem cells.
• stems cells In humans there are two main types of stems cells: embryonic stem cells and adult stem cells. In a developing embryo stem cells differentiate into all types of cells, thereby creating specialized tissues, organs, and systems. In an adult human, stem cells are involved in the normal turnover of organs such as blood and skin.
• Hematopoietic stem cells are used to treat blood and immune system diseases because they can differentiate into red blood cells, white blood cells, and platelets.
• some stem cell transplants have been performed for patients with genetic or metabolic diseases. Indeed, to date more than 80 different diseases have been treated using stem cell transplants.
• the National Cord Blood Program there were over 15,000 through the end of 2009.
• the National Marrow Donor Program estimates that there will be 10,000 cord blood transplants per year by 2015, up from 2,000 per year in 2006.
• stem cells In addition to known treatments involving stem cells, research continues into the promise of many potential future applications. Indeed, the ability of stem cells to differentiate into other types of cells holds significant promise for treating some of the world's most common diseases including heart disease, diabetes, stroke, hearing loss, blood disorders, Parkinson's disease, and Alzheimer's disease, just to name a few.
• Umbilical cord blood - blood which remains in the placenta and umbilical cord after childbirth - is one of the most common sources of stem cells. Since cord blood is collected from the placenta, which is normally discarded, the collection process is safe for both the mother and the newborn.
• Cord blood is obtained by syringing out the placenta through the umbilical cord shortly after childbirth, after the cord has been detached from the newborn. The retrieved blood can then be frozen and stored indefinitely.
• the placenta is a better source of stem cells, since it can contain up to ten times more stem cells than cord blood. Still, even when blood is retrieved from both the umbilical cord and placenta using current collection methods, the amount of stem cells is often not suitable to treat an adult patient.
• a method for stem cell collection comprising:
• the collection solution comprises: (a) a placental preservative base; (b) a stem cell releasing agent; (c) an antibiotic; and (d) a vasodilator.
• the stem cell releasing agent comprises 1,1 '-[1,4-
• Phenylenebis(methylene)]bis[l,4,8,l l-tetraazacyclotetradecane] also known as AMD3100
• AMD3100 Phenylenebis(methylene)]bis[l,4,8,l l-tetraazacyclotetradecane]
• the antibiotic is an aminoglycoside such as gentamicin, and can be present at a concentration of approximately 10 mg per 100 ml.
• the vasodilator is one or more of papaverine nitroglycerine, a Ca 2+ channel blocker, an opium alkaloid, and combinations thereof.
• the vasodilator can be present at a concentration of approximately 100 mg per 100 ml.
• the placental preservative base comprises: (i)
• the placental preservative base can comprise, for example, the following: (i) NaCl at a concentration of approximately 4.3 g/L; (ii) KCl at a concentration of approximately 0.45 g/L; (iii) D-Glucose at a concentration of approximately 1 g/L; (iv) citric acid at a concentration of approximately 2.2 g/L; (v) adenine at a concentration of approximately 0.25 g/L; (vi) histidine at a concentration of approximately 4.2 g/L; (vii) glutamate at a concentration of approximately 1.9 g/L; (viii) glutathione at a concentration of approximately 0.92 g/L or 3 mM; and (ix) N-acetyl-L- cysteine at a concentration of approximately 0.016 g/L.
• the collection solution further comprises a synthetic colloidal material, preferably creating colloidal osmotic pressure.
• the synthetic colloidal material can comprise a hydroxyethyl starch.
• the collection solution further comprises an anticoagulant such as heparin.
• the collection solution further comprises a carbohydrate such as trehalose.
• the method comprises one or more of the additional steps in no particular order: collecting the perfusate; transporting the perfused placenta; isolating the stem cells from the perfusate; and/or combining the collected exsanguinated blood and the collected perfusate.
• a stem cell collection solution comprising: (i) a placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; and (iv) a vasodilator.
• the stem cell collection solution can comprise, for example, the following: ⁇ , - [ 1 ,4-Phenylenebis(methylene)]bis[ 1 ,4,8, 11-tetraazacyclotetradecane] (AMD3100); gentamicin; and/or papaverine.
• the placental preservative base can comprises, for example, the following: (i) NaCl; (ii) KCl; (iii) D-Glucose; (iv) citric acid; (v) adenine; (vi) histidine; (vii) glutamate; (viii) glutathione; and/or (ix) N-acetyl-L-cysteine.
• the stem cell collection solution can further comprises an anticoagulant.
• a stem cell collection kit comprising a stem cell collection solution which comprises: a placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; and (iv) a vasodilator.
• the stem cell releasing agent comprises l, -[l,4-Phenylenebis(methylene)]bis[l,4,8,l 1-tetraazacyclotetradecane] (AMD3100), the antibiotic comprises gentamicin, the vasodilator comprises papaverine.
• the placental preservative base comprises: (i) NaCl; (ii) KC1; (iii) D-Glucose; (iv) citric acid; (v) adenine; (vi) histidine; (vii) glutamate; (viii) glutathione; and/or (ix) N-acetyl-L-cysteine.
• FIG. 1 is a flowchart of a method of stem cell collection according to one embodiment.
• FIG. 2 is a flowchart of a method of umbilical cord segment collection according to one embodiment.
• FIG. 1 a flowchart of a method of stem cell collection according to one embodiment of the present invention.
• step 10 is the primary cord blood collection procedure. This is accomplished using any of a variety of known cord blood collection methods, or any method capable of collecting the excess cord blood from the umbilical cord and/or placenta shortly after childbirth.
• the cord blood can be collected using the following procedure: (i) deliver the baby vaginally or via cesarean section; (ii) clamp the umbilical cord at both the mother's and the child's side and cut the cord at a location between the clamps; (iii) insert a tube or needle into the umbilical vein to collect the blood via gravity; and (iv) ship the blood off for processing and dispose of the placenta.
• the cord blood is collected after delivery of the baby(ies) but before delivery of the placenta.
• the placenta is typically disposed and/or destroyed upon completion of the typical cord blood collection procedure. As such, these methods do not proceed past step 10 of the present method.
• the placenta is filled with the cord blood collection solution.
• the cord blood collection solution comprises one or more of the following solutions.
• the cord blood collection solution comprises the following: (i) placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; and (iv) a vasodilator.
• the cord blood collection solution comprises the following: (i) placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; (iv) vasodilator; and (v) synthetic colloidal material.
• the cord blood collection solution comprises the following: (i) placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; (iv) a vasodilator; (v) a synthetic colloidal material; and (vi) an anticoagulant.
• the cord blood collection solution comprises the following: (i) placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; (iv) a vasodilator; (v) a synthetic colloidal material; and (vi) a carbohydrate.
• the stem cell releasing agent can be any agent capable of causing stem cells to release from normal storage sites into the blood.
• the stem cell releasing agent is AMD3100 (l,l'-[l,4-Phenylenebis(methylene)]bis[l,4,8,l l- tetraazacyclotetradecane], also known by the recommended International Nonproprietary Name plexifor, by the trade name MOZOBIL ® , and as JM 3100), preferably at a concentration of approximately 3-10 mg per 100 ml, although other concentrations are possible.
• the AMD3100 causes, among other conditions, the release of stem cells from the placenta blood vessel walls, resulting in substantially higher amounts of stem cells to be collected via the process described herein.
• the antibiotic can be, for example, any agent capable of providing bactericidal properties against a range of human bacterial infections, while being substantially non-toxic to humans.
• One such antibiotic is Gentamicin, which is preferably at a concentration of approximately 10 mg per 100 ml in this embodiment, although other concentrations are possible.
• the vasodilator can be any agent capable of dilating the blood vessels of the umbilical cord and placenta.
• One such vasodilator is papaverine, which is preferably at a concentration of approximately 100 mg per 100 ml in this embodiment, although other concentrations are possible.
• Other vasodilators include but are not limited to nitroglycerine, Ca 2+ channel blockers, and other opium alkaloids, for example.
• the synthetic colloidal material can be any synthetic colloidal material, including those intended to create colloidal osmotic pressure and prevent the expansion of the extracellular space in tissues.
• the synthetic colloidal material can include dextrans, hydroxyethyl starch (“HES”) such as Hespan ® , and polyethylene glycol.
• HES hydroxyethyl starch
• the synthetic colloidal material is Hespan at a concentration of approximately 4 to 8%, and preferably 6% (6g/100ml), although other concentrations are possible.
• the anticoagulant can be any compound capable of preventing the coagulation of blood and associated or similar fluids.
• the anticoagulant is the sulfated glycosaminoglycan heparin at a concentration of approximately 400 - 600 units per 200 ml, although other concentrations are possible.
• the carbohydrate can be any known natural or synthetic carbohydrate.
• the carbohydrate is the disaccharide trehalose at a concentration of 2- 6%, and preferably at 4% (4g/100ml), although other concentrations are possible.
• the placental preservative base can be any solution capable of preserving the placenta during shipping and short- and/or long-term storage.
• the placental preservative base comprises the following: (i) NaCl (4.3 g/L or 74 mM); (ii) KC1 (0.45 g/L or 6 mM); (iii) D-Glucose (1 g/L or 5.6 mM); (iv) citric acid anhydrous (2.2 g/L or 15 mM); (v) adenine free base (0.25 g/L or 1.85 mM); (vi) histidine (4.2 g/L or 20 mM); (vii) glutamate (1.9 g/L or 13 mM); (viii) glutathione (0.92 g/L or 3 mM; and/or (ix) N-Acetyl L-Cysteine (also known as acetylcysteine or
• the target electrolyte and glucose profile of the placental preservative base comprises: (i) approximately 0 to 250 mM Na + , with a preferred embodiment comprising approximately 125 to 150 mM Na + ; (ii) approximately 0 to 25 mM K + , with a preferred embodiment comprising approximately 5 to 7 mM K + ; (iii) approximately 0 to 250 mM CI " , with a preferred embodiment comprising approximately 50 to 150 mM CI " ; (iv) 0 to 10 mM glucose, with a preferred embodiment comprising approximately 5 mM glucose; (v) and osmolarity of 100 to 400 mOsm/kg, with a preferred embodiment comprising approximately 280 to 300 mOsm/kg.
• the cord blood collection solution is allowed to fill the placenta via gravity through a needle or tube inserted into the umbilical vein and/or other locations in the placenta.
• the placenta contains a sufficient amount of cord blood collection solution, decided by a variety of factors including the predetermined amount of solution provided for collection, the size of the placenta, and/or prescribed collection procedure, among many others, the umbilical cord is once again clamped shut such that the injected solution remains inside the placenta.
• the placenta is processed for shipping to a processing/collection center.
• This can include, among other things, processing the umbilical cord to ensure clamping, placing the placenta in a container suitable for short- or long-term shipping or storage, and/or freezing the placenta. Together with the primary cord blood collection, the placenta is then shipped or delivered to the processing/collection center.
• the processing/collection center may be the same institution or near the same institution as the baby delivery center.
• the placenta undergoes processing at the processing/collection center.
• the cord blood collection solution is collected from the placenta at this step.
• This "secondary collection” is then processed to concentrate the mononuclear cell content and is then cryogenically stored together, but in separate containers, with the primary cord collection.
• the two collections may or may not be combined prior to storage depending upon a variety of factors, including but not limited to storage limitations and known or possible future uses of the stem cells, among others.
• the placenta can be cryogenically stored as well, or can be disposed of once the procedure is complete.
• the cord blood collection solution is allowed to drain from the placenta via gravity through a needle or tube inserted into the umbilical vein and/or other locations in the placenta.
• the umbilical cord is undamped to allow the egress of fluid, and any drained fluid is then collected.
• the placenta is cryogenically stored prior to the secondary collection.
• the injected cord blood collection solution is stored inside the placenta until it is needed, or until it can be more properly processed.
• Umbilical cord segment collection is a growing field, as these segments contain materials that can be cultivated and manipulated for beneficial uses. For example, stem cells can be harvested from the segment and used to grow a sufficiently large collection of stem or differentiated (fully or partially) cells which themselves can be used immediately or stored for future possible use.
• step 20 of the method is the collection and cleaning of the umbilical cord.
• the umbilical cord is harvested and can be cleaned using any suitable technique for sterilizing the exterior surface of the cord to prevent contamination.
• a segment of the umbilical cord of a desired length is cut out.
• the desired length may be a 1-inch long segment of the cord, or may be longer or shorter depending upon the downstream processing needs.
• the umbilical cord is cut using sterile tools and techniques.
• the cut segment is placed into the umbilical cord storage/collection solution.
• the solution can be any of the previous solutions known in the art or any of the novel solutions described herein.
• the collector has a tube pre-filled with the solution and simply places the umbilical cord segment into the tube (thereby immersing it in the solution), perhaps with mixing by shaking or inverting the tube several times.
• the tube is sized to fit the exact size of the umbilical cord segment and the solution. Once in the solution and in the tube, the umbilical cord segment is secure and the cells and tissues within that umbilical cord segment are well- maintained.
• the umbilical cord segment is ready for shipment or deliver to a collection or storage center at step 26 of the method.
• the segment can be immediately processed at the center, or can be stored for processing in the future.
• the system may comprise all of some of the components needed to procure cells from a placenta and/or collect umbilical cord segments.
• the system may comprise a device for placental harvesting and stabilization during processing of the placenta according to one or more of the methods disclosed herein.
• the system may also comprise, for example, clamps, needles, tubing, scissors, scalpel, or other components to facilitate the procurement of cells from a placenta.
• many other devices known in the art for harvesting, stabilizing, holding, and/or transporting a placenta would be suitable for the system.
• the system may also comprise a transportation element, such as a sleeve, container, envelope, or other element comprising space for transportation of the placenta to a collection and/or storage site.
• a transportation element such as a sleeve, container, envelope, or other element comprising space for transportation of the placenta to a collection and/or storage site.
• the transport element may also comprise space for transporting all of some of the collected liquid/cells from the placenta to the collection and/or storage site.
• the system comprises cord blood collection solution, according to an embodiment.
• the components may be stored as a single solution, or one or more of the components may be stored separately and require mixing with the remainder of the solution prior to collection.
• the cord blood collection solution comprises a container, bag, or other holding element containing the following: (i) placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; and (iv) a vasodilator.
• the cord blood collection solution may also comprise a synthetic colloidal material, an anticoagulant, and/or a carbohydrate.
• the placental preservative base comprises one or more of the following: NaCl, KC1, D-Glucose, citric acid anhydrous, adenine free base, histidine, glutamate, glutathione, and/or N- Acetyl L-Cysteine.
• kits for the procurement of placental stem cells and/or umbilical cord segments may comprise all of some of the components needed to procure cells from a placenta and/or collect umbilical cord segments.
• the kit may comprise a device for placental harvesting and stabilization during processing of the placenta according to one or more of the methods disclosed herein.
• the kit may also comprise, for example, clamps, needles, tubing, scissors, scalpel, or other components to facilitate the procurement of cells from a placenta and/or the collection of umbilical cord segments.
• many other devices known in the art for harvesting, stabilizing, holding, and/or transporting a placenta and/or collect umbilical cord segments would be suitable for the kit.
• the kit may also comprise a transportation element, such as a sleeve, container, envelope, or other element comprising space for transportation of the placenta to a collection and/or storage site.
• a transportation element such as a sleeve, container, envelope, or other element comprising space for transportation of the placenta to a collection and/or storage site.
• the transport element may also comprise space for transporting all of some of the collected liquid/cells from the placenta to the collection and/or storage site.
• the kit comprises cord blood collection solution, according to an embodiment.
• the cord blood collection solution comprises a container, bag, or other holding element containing the following: (i) placental preservative base; (ii) a stem cell releasing agent; (iii) an antibiotic; and (iv) a vasodilator.
• the cord blood collection solution may also comprise a synthetic colloidal material, an anticoagulant, and/or a carbohydrate.
• the placental preservative base comprises one or more of the following: NaCl, KC1, D-Glucose, citric acid anhydrous, adenine free base, histidine, glutamate, glutathione, and/or N- Acetyl L-Cysteine.

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• 2013
  • 2013-01-30 WO PCT/US2013/023868 patent/WO2013110094A1/fr not_active Ceased

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