CN106719599A - It is a kind of to reduce the method that Cryopreserved histoorgan ice crystal is damaged - Google Patents

Abstract

The invention provides a method for reducing the ice crystal damage of cryopreserved tissues and organs. The method for processing the cryopreserved organs provided by the present invention uses stem cell conditioned medium perfusion before the organs are to be frozen, which improves the survival of tissue cells. state, increased the anti-apoptotic ability, and reduced the oxidative stress damage of the cells in vitro; immediately after resuscitation, the complete medium containing fresh mesenchymal stem cells was used for post-perfusion treatment, and the damage caused by the cryopreservation process The active repair of cells damaged by cryoprotectant toxicity and osmosis can effectively reduce the rate of apoptosis and reduce the damage of tissue structure.

Description

A method for reducing ice crystal damage of cryopreserved tissues and organs

technical field

The invention belongs to the field of cryopreservation of organs and tissues in clinical medicine, and discloses a method of perfusing mesenchymal stem cells and their secretion products before and after cryopreservation of organs and tissues, so as to reduce cell apoptosis and ice crystal damage to cell membranes. A method of preprocessing and postprocessing.

Background technique

Organ transplantation is the ultimate means of treating severe organ failure, but there are always differences in time and space between donor donation and recipient preparation. Therefore, the further development of safer and long-term organ cryopreservation-resuscitation methods has become an urgent problem to be solved .

The cryopreservation process of biological tissue includes perfusion of cryoprotectant, programmed cooling, cryogenic storage, resuscitation and thawing, and replacement and cleaning of cryoprotectant. Each step affects the survival rate of cells in the organ after freezing and the maintenance of tissue structure and function. In 1972, Mazur et al. first proposed a two-factor hypothesis for freezing injury from experiments on cryopreservation of Chinese hamster tissue culture cells. One is the solute damage effect, which means that the cooling rate is too slow, resulting in an increase in the concentration of extracellular solutes. The second is the mechanical damage effect, which means that the cooling rate is too fast, and the ice crystals formed in the cells directly damage the membrane structure of the cells.

In order to reduce the damage to cells caused by cryopreservation, vitrification is now commonly used to increase the viscosity of the liquid and reduce the formation of internal crystals during freezing and solidification. The realization of vitrification of the solution mainly depends on increasing the cooling rate and increasing the concentration of the solution, which requires the use of cryoprotectants. However, the cryoprotectant itself can also cause damage to the cells. High concentrations of the protective agent can cause toxic effects on cells and tissues, and the osmotic pressure difference between the inside and outside of the cell membrane can also be caused during the elution process after rewarming, forming osmotic damage. Therefore, the focus of current cryogenic research is to reduce the damage of cryoprotectants during vitrification. The direction of existing experiments mainly includes improving the introduction and elution methods, using the "continuous introduction method"; "negative pressure impregnation technology" to speed up the penetration of protective agents; and the compounding technology of using different types of cryoprotectants to make them diluted with each other. However, in addition to their own characteristics, cryoprotectants have different protective and toxic effects in different cell tissues. An organ is not a simple collection of various cells. As a composite tissue with a complex structure, the difference in cooling rate caused by the uneven temperature distribution of each part during the cooling process and the different sensitivity of various cells to cryoprotectants make cryoprotection difficult. The difficulty of saving is greatly increased. Therefore, it is extremely important to find new cryoprotection methods for tissue cells and methods for repairing damage.

Contents of the invention

In order to solve the above technical problems, the present invention provides a method of pre-perfusion and post-perfusion in tissues using umbilical cord mesenchymal stem cells and their conditioned medium in the process of freezing and storing organs, which can effectively improve the self-repair of tissues The ability to effectively increase the proportion of living cells in the tissue after resuscitation, so that tissues and organs can better recover their functions after rewarming.

The organ cryopreservation method related to the present invention comprises the following steps:

1) Connect the organ to be frozen to a gradient concentration mixed program-controlled cooling organ perfusion apparatus pre-cooled to 4°C, and perfuse the mesenchymal stem cell conditioned medium at a constant temperature of 25°C for 30-60 minutes;

The mesenchymal stem cell conditioned medium is a kind of mesenchymal stem cells in fat, umbilical cord, umbilical cord blood or bone marrow that have been passaged for 5 generations, and grow to 70-80% confluence 24 hours after passage, and replace with serum-free mesenchymal stem cells. Stem cell culture medium, the culture supernatant collected after 48 hours, centrifuged at 1000~3000g for 10~30min, and the supernatant was obtained after filtration with a 0.1µm~0.22µm microporous membrane;

2) Then perfuse the organ with a cryoprotectant pre-cooled to 4°C for 30 minutes;

3) Put the processed organs into a freezer bag, seal it and place it in a program-controlled cooling device, perform programmed cooling according to the set cooling program, and put it into liquid nitrogen for storage after the temperature drops to -80°C;

The cooling program of the program-controlled cooling instrument is set as ① balance at 4°C, ② drop to 0°C at a rate of 1°C/min, and keep it for 10 minutes, ③ drop to -18°C at a rate of 2°C/min, and keep it for 15 minutes, ④ with 2°C/min to -45°C, keep for 15 minutes, ⑤ drop to -90°C at 5°C/min, and keep for 5 minutes;

4) When resuscitating the organs, take the frozen organs out of liquid nitrogen and place them in a 37°C water bath, shaking them constantly;

5) Perfuse again after complete melting, the perfusion solution is conditioned medium containing mesenchymal stem cells at 12-25°C, and the perfusion time is 30min-60min;

The processed organs can be directly transplanted.

The conditioned medium of the mesenchymal stem cells has a cell density of 1×10 ⁷ /L and a perfusion rate of 1.5 ml/min.

The treatment method of this cryopreserved organ provided by the present invention uses stem cell conditioned medium perfusion before the organ is to be frozen, which improves the survival state of tissue cells, increases the anti-apoptotic ability, and reduces the occurrence of cell death in vitro. Oxidative stress damage; Immediately after resuscitation, post-perfusion treatment with complete medium containing fresh mesenchymal stem cells is used to actively repair cells that have been damaged by cryoprotectant toxicity and osmotic damage during cryopreservation, which can effectively reduce cell apoptosis. Mortality rate, reduce the destruction of organizational structure.

Description of drawings

Fig. 1. is the flowchart of the deep cryopreservation method for organs provided by the present invention;

Figure 2. Renal artery catheterization and ligation;

Figure 3. HE staining results of specimens in each group;

A is HE staining of fresh rat kidney paraffin section (40×);

B is HE staining of fresh rat kidney paraffin section (200×);

C is HE staining (40×) of kidney paraffin section of rats in cryopreserved control group;

D is HE staining (200×) of kidney paraffin section of rats in cryopreserved control group;

E is HE staining of kidney paraffin section of rats in MSC perfusion group (40×);

F is HE staining of kidney paraffin section of rats in MSC perfusion group (200×);

Figure 4. Paller's score of renal tubules of rats in each experimental group;

Figure 5. TUNEL staining results of specimens in each group;

A, B. TUNEL staining of fresh rat kidney paraffin section (200×);

C, D. TUNEL staining (200×) of kidney paraffin sections of frozen-preserved rats in the control group;

E, F. TUNEL staining (200×) of paraffin sections of kidneys of rats perfused with MSCs.

detailed description

In order to clarify the purpose, technical solution and advantages of the present invention, the technical solution of the present invention will be clearly and completely described below by taking rat kidney as an example.

Example 1

Cryopreservation and Recovery of Rat Kidney

Adipose-derived mesenchymal stem cells cultured to the 4th to 6th passages were taken, and fresh medium was replaced after passage, and the culture supernatant was collected after 48 hours for use.

SD rats aged 8-12 weeks were selected and anesthetized by intraperitoneal injection of chloral hydrate. The abdominal cavity was opened, and the kidney was removed, keeping the integrity of the renal artery and vein structure.

The removed kidneys were rinsed in 4°C physiological saline, and then placed in the same pre-cooled stem cell conditioned medium.

Through renal artery cannulation and silk thread fixation, after ligation of the renal artery branch, it was connected to a program-controlled cooling organ perfusion instrument pre-cooled to 4°C, and the quantitative infusion pump was started to perfuse the stem cell conditioned medium into the kidney at a rate of 1.5ml/min. 60min (Figure 2).

The perfusate was changed to cryoprotectant, and perfusion was performed in the same way.

Put the perfused kidney into a freezing bag and place it in a programmed cooling device. The program is set to ① balance at 4°C, ② drop to 0°C at a rate of 1°C/min, and keep it for 10 minutes, and ③ keep at a rate of 2°C/min. Lower to -18°C and keep for 15 minutes, ④ drop to -45°C at a rate of 2°C/min, and keep for 15 minutes, and ⑤ drop to -90°C at a rate of 5°C/min, and keep for 5 minutes. Then put the cryopreservation bag into liquid nitrogen.

After one week of frozen storage, the frozen kidneys were taken out, placed in a 37°C water bath to thaw, and shaken constantly to make the temperature distribution even.

The adipose-derived mesenchymal stem cells cultured to passage 4-6 were digested with trypsin, and resuspended in conditioned medium precooled to 4°C to adjust the cell concentration to 1×10 ⁷ /L.

After the organ was completely melted in the water bath, it was transferred into the stem cell conditioned medium, and the renal artery cannula was connected to the perfusion apparatus again, and the stem cell suspension described in step 8 was used for perfusion at a rate of 1.5ml/min for 30min.

Example 2

Determination of viability after thawing of cryopreserved organs

In the cryopreservation experiment, the rat kidneys were divided into three groups, namely:

Fresh group: Immediately fix the kidneys in 4% paraformaldehyde after taking them out, without freezing them.

Control group: cryopreserved according to the general cryopreservation method without stem cell treatment.

Experimental group: cryopreserved according to the method of the present invention.

After one week of cryopreservation, the organs of each group were revived, and their structural integrity and cell viability were measured.

Morphological analysis

Tissue Paraffin Section Fabrication

Paraffin embedding and sectioning: the kidneys of each group were cut along the coronal plane through the renal artery, and half of the tissues were fixed in 4% paraformaldehyde. The other half of the tissue was used for molecular biology testing. The fixed tissues were dehydrated, transparentized, soaked in wax, and embedded to make paraffin tissue blocks, which were sliced coronally on a paraffin microtome with a thickness of 4um.

Hematoxylin-eosin staining (HE staining)

Dewaxing and hydration: bake paraffin sections at 60°C for 30 minutes, then add xylene for 20 minutes×2, absolute ethanol for 10 minutes×2, 95% ethanol for 5 minutes×2, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes , PBS 5min×3.

Staining and differentiation: Immerse the slices in hematoxylin staining solution for 10 minutes, drop 1% hydrochloric acid alcohol solution for 5 seconds, rinse with tap water for 10 minutes to turn blue.

Counterstaining: immerse in 0.5% eosin staining solution for 2-5 minutes, rinse with PBS for 10 minutes.

Dehydration and sealing: conventional gradient ethanol dehydration, immerse the stained sections in 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 10 min×2, absolute ethanol for 10 min×2, xylene for 10 min×2, neutral gum for sealing .

Observe and take pictures under a light microscope.

Morphological analysis:

I. After cryopreservation and resuscitation, the kidneys of the control group and MSC perfusion group can be seen in the renal cortex area with acute degeneration and necrosis of the proximal renal tubules as the main feature; Partial cell vacuolar degeneration and swelling rupture, nuclei condense, rupture or even disappear, part of the basement membrane is exposed or incomplete, exfoliated epithelial cells can be seen in the lumen, cell debris is filled in the lumen of many small tubules, and the lumen is narrowed or blocked , Diffuse interstitial edema. Glomeruli showed no obvious pathological changes (Figure 3).

II. Paller’s score for renal tubular damage: select 10 high-power microscope fields of view for each specimen, and score the 10 renal tubules with the most serious lesions. A total of 100 renal tubules with lesions are counted. Scoring standard: renal tubules are obviously dilated 1 point for flat renal tubular epithelial cells; 1 point for brush border shedding; 1-2 points for vacuolar degeneration of renal tubular epithelial cells; 1 point for interstitial edema; 1-2 points for lumen obstruction or cast; 1 point for tubular epithelial cell necrosis -2 points; 1-2 points for renal tubular epithelial cell membrane vesicle formation.

The results of Paller's score for renal tubule injury showed that the renal tubules were damaged during cryopreservation in the control group and the experimental group, but the score of the experimental group treated with the method of the present invention was lower than that of the cryopreservation control group (Figure 4).

detection

① The dewaxing and hydration process is as described above

② Immerse the slices in 3% hydrogen peroxide and incubate at room temperature for 10 minutes. Rinse with PBS for 2min×3;

③ Immerse the slices in 100mM Tris/HCl containing 10µg/L proteinase K, digest at 37°C for 20min, wash with PBS for 5min×3;

④ Shake off the excess liquid on the slice, and add 100µl TUNEL reaction solution dropwise. Negative controls used distilled water instead of TdT enzyme. Incubate at 37°C in the dark for 60 minutes;

⑤ Rinse with PBS for 5min×3, mix DAPI staining solution with water-based mounting medium and mount the slide;

⑥ Observation under the microscope, randomly select 5 fields of view from each section to take pictures and evaluate the number of apoptotic cells.

The morphological changes of nuclear chromatin in the process of cell apoptosis can be divided into three phases: in phase I, the nucleus is corrugated or folded, and part of the chromatin is condensed; in phase IIa, the chromatin of the nucleus is highly condensed and marginalized; in phase IIb The nuclei of the cells at this stage are cleaved into fragments, producing apoptotic bodies. Apoptotic cells were stained with TUNEL and looked green under a laser microscope.

The results showed that the number of apoptotic cells in organs treated by the method of the present invention was less than that in the control group (Figure 5).

Claims (2)

1. A method for reducing cryopreserved tissue and organ ice crystal damage, characterized in that, comprising the following steps: 1) Connect the organ to be frozen to a gradient concentration mixed program-controlled cooling organ perfusion apparatus pre-cooled to 4°C, and perfuse the mesenchymal stem cell conditioned medium at a constant temperature of 25°C for 30-60 minutes; The mesenchymal stem cell conditioned medium is a kind of mesenchymal stem cells in fat, umbilical cord, umbilical cord blood or bone marrow that have been passaged for 5 generations, and grow to 70-80% confluence 24 hours after passage, and replace with serum-free mesenchymal stem cells. Stem cell culture medium, the culture supernatant collected after 48 hours, centrifuged at 1000~3000g for 10~30min, and the supernatant was obtained after filtration with a 0.1µm~0.22µm microporous membrane; 2) Then perfuse the organ with a cryoprotectant pre-cooled to 4°C for 30 minutes; 3) Put the processed organs into a freezer bag, seal it and place it in a program-controlled cooling device, perform programmed cooling according to the set cooling program, and put it into liquid nitrogen for storage after the temperature drops to -80°C; The cooling program of the program-controlled cooling instrument is set as ① balance at 4°C, ② drop to 0°C at a rate of 1°C/min, and keep it for 10 minutes, ③ drop to -18°C at a rate of 2°C/min, and keep it for 15 minutes, ④ with 2°C/min to -45°C, keep for 15 minutes, ⑤ drop to -90°C at 5°C/min, and keep for 5 minutes; 4) When resuscitating the organs, take the frozen organs out of liquid nitrogen and place them in a 37°C water bath, shaking them constantly; 5) Perfuse again after complete melting, the perfusion solution is conditioned medium containing mesenchymal stem cells at 12-25°C, and the perfusion time is 30min-60min; The processed organs can be directly transplanted. 2 . The method according to claim 1 , wherein the conditioned medium for mesenchymal stem cells has a cell density of 1×10 ⁷ /L and a perfusion rate of 1.5 ml/min.