CN106719599B - Method for reducing ice crystal damage of deep low temperature frozen tissue and organ - Google Patents

Abstract

The invention provides a method for reducing ice crystal damage of cryopreserved tissues and organs, and the method for treating the cryopreserved organs provided by the invention uses stem cell conditioned medium to perfuse before the organs are frozen, which improves the survival of tissue cells state, increased the anti-apoptotic ability, and reduced the oxidative stress damage of the cells after ex vivo; the complete medium containing fresh mesenchymal stem cells was used for post-perfusion treatment immediately after resuscitation, and the cells were subjected to post-perfusion treatment during cryopreservation. Active repair of cells damaged by cryoprotectant toxicity and osmotic damage can effectively reduce the rate of apoptosis and reduce the damage to 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 the use of mesenchymal stem cells and their secreted products to perfuse before and after cryopreservation of organs and tissues, thereby reducing cell apoptosis and reducing ice crystal damage to cell membranes. methods of preprocessing and postprocessing.

Background technique

Organ transplantation is the ultimate treatment for severe organ failure, but there are always differences in time and space between donor donation and recipient preparation. Therefore, further development of safer and longer-lasting 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, thawing and replacement of cryoprotectant. Each step affects the viability of cells in the frozen organ and the maintenance of tissue structure and function. In 1972, Mazur et al. first proposed the two-factor hypothesis of freezing injury from the experiment of 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 formation of ice crystals in the cells directly damages the membrane structure of the cells.

In order to reduce the damage of cryopreservation to cells, vitrification is now often used to increase the viscosity of the liquid and reduce the formation of internal crystals during the freezing and solidification process. Vitrification of the solution is mainly achieved by 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 protectant can cause toxic effects on cells and tissues. During the elution process after rewarming, the osmotic pressure difference between the inside and outside of the cell membrane will also be caused, resulting in osmotic damage. Therefore, the current focus of cryogenic research is to reduce the damage of cryoprotectants during vitrification. The main directions of the existing experiments are to improve the introduction and elution methods, using the "continuous introduction method"; the "negative pressure dipping technology" to accelerate the penetration of the protective agent; the compound technology of using different types of cryoprotectants to dilute each other. However, in addition to its own characteristics, cryoprotectants have different protective and toxic effects in different cell tissues. Organs are not a simple collection of various cells. As a complex tissue with a complex structure, the difference in the cooling rate caused by the uneven temperature distribution of each part during the cooling process and the different sensitivities of various cells to cryoprotectants make them cryogenically frozen. The difficulty of saving is greatly increased. Therefore, it is extremely important to find new methods of cryoprotection of tissue cells and methods of repairing damage.

SUMMARY OF THE INVENTION

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

The cryopreservation method for organs involved in 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 min;

The mesenchymal stem cell conditioned medium is the mesenchymal stem cells of one of fat, umbilical cord, umbilical cord blood or bone marrow within 5 generations of passage, and grows to 70-80% confluence after 24 hours of passage, and replaces serum-free mesenchymal stem cells. Stem cell culture medium, the culture supernatant collected after 48 hours was centrifuged at 1000~3000g for 10~30min, and the supernatant was collected and filtered with a 0.1µm~0.22µm microporous membrane;

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

3) Put the processed organs into freezer bags, seal them, and place them in a program-controlled cooling device. Perform programmed cooling according to the set cooling program. After the temperature drops to -80°C, put them into liquid nitrogen for storage;

The program-controlled cooling device cooling program is set as ① 4 ℃ balance, ② drop to 0 ℃ at a speed of 1 ℃/min, and keep for 10 min, ③ drop to -18 ℃ at a speed of 2 ℃/min, keep for 15 min, ④ with a 2°C/min speed to -45°C, hold for 15min, ⑤ Then drop to -90°C at 5°C/min speed, hold for 5min;

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

5) After complete thawing, perform perfusion again. The perfusion solution is conditioned medium containing mesenchymal stem cells at 12-25°C, and the perfusion time is 30min-60min;

The processed organ can be directly transplanted.

In the conditioned medium containing mesenchymal stem cells, the cell density is 1×10 ⁷ cells/L, and the perfusion speed is 1.5 mL/min.

The processing method of the cryopreserved organ provided by the present invention uses stem cell conditioned medium to perfuse 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 damage after being isolated from the body. Oxidative stress damage; post-perfusion treatment with complete medium containing fresh mesenchymal stem cells immediately after resuscitation can actively repair cells that have been subjected to cryoprotectant toxicity and osmotic damage during cryopreservation, which can effectively reduce cell apoptosis death rate and reduced tissue damage.

Description of drawings

Fig. 1. is the flow chart of the cryopreservation method of organs provided by the present invention;

Figure 2. Renal artery cannulation and ligation;

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

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

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

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

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

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

F is the HE staining of paraffin section of rat kidney 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 each group of specimens;

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

C, D. TUNEL staining (200×) of paraffin sections of the kidneys of rats in the cryopreserved control group;

E, F. TUNEL staining (200×) of renal paraffin sections in MSC perfusion group.

Detailed ways

In order to clarify the purpose, technical solution and advantages of the present invention, the following takes the rat kidney as an example to clearly and completely describe the technical solution of the present invention.

Example 1

Cryopreservation and resuscitation of rat kidneys

The adipose-derived mesenchymal stem cells cultured to the 4th to 6th generation were taken, replaced with fresh medium 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, taking care to keep the renal arteries and veins intact.

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

The renal artery was cannulated and fixed with silk thread. After ligating the renal artery branch, it was connected to a programmed cooling organ perfusion apparatus pre-cooled to 4°C. The quantitative infusion pump was started, and the stem cell conditioned medium was perfused into the kidney at a rate of 1.5 mL/min for a period of time. 60min (Figure 2).

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

The perfused kidney was put into a freezing bag and placed in a programmed cooling device. The program was set to ① 4°C to balance, ② to drop to 0°C at a speed of 1°C/min, and keep it for 10 min, and ③ to set a speed of 2°C/min. Drop to -18°C, hold for 15 minutes, ④ reduce to -45°C at a rate of 2°C/min, hold for 15 minutes, and ⑤ drop to -90°C at a rate of 5°C/min and hold for 5 minutes. The cryopreservation bag was then dropped into liquid nitrogen.

After one week of cryopreservation, the cryopreserved kidneys were taken out, thawed in a 37°C water bath, and shaken at all times to distribute the temperature evenly.

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

After the organ was completely thawed in a water bath, it was transferred into the stem cell conditioned medium, the renal artery cannula was connected to the perfusion apparatus again, and the stem cell suspension described in step 5 was used for perfusion at a rate of 1.5 mL/min for 30 min.

Example 2

Determination of the viability of cryopreserved organs after resuscitation

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

Fresh group: The rat kidneys were fixed in 4% paraformaldehyde immediately after taking them, without freezing.

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

Experimental group: cryopreserved according to the method described in the present invention.

Organs in each group were recovered after one week of cryopreservation, and their structural integrity and cell viability were determined.

Morphological analysis

Preparation of tissue paraffin sections

Paraffin embedding and sectioning: The kidneys of each group were incised along the coronal plane through the renal artery, and half of the tissues were placed in 4% paraformaldehyde for fixation. The other half of the tissue is used for molecular biology testing. The fixed tissue was dehydrated, transparent, immersed in wax, and embedded to make paraffin tissue blocks, which were serially sectioned on a paraffin microtome with a thickness of 4um.

Hematoxylin-eosin staining (HE staining)

Dewaxing and hydration: bake the paraffin sections at 60°C for 30 minutes, then put them into 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 differentiation: immerse the sections in hematoxylin staining solution for 10min, drop 1% hydrochloric acid alcohol solution for 5s, and rinse with tap water for 10min.

Counterstaining: immersed in 0.5% eosin staining solution for 2-5min, rinsed with PBS for 10min.

Dehydration and mounting: Dehydrate with conventional gradient ethanol, immerse the stained sections in 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 10 min × 2, anhydrous ethanol for 10 min × 2, xylene for 10 min × 2, and mount with neutral gum .

Observe and take pictures under a light microscope.

Morphological analysis:

I. After cryopreservation and resuscitation, the kidneys of the control and MSC perfusion groups showed pathological changes characterized by acute degeneration and necrosis of proximal tubules in the renal cortex; Some cells were vacuolated, swollen and ruptured, nuclei were concentrated, ruptured or even disappeared, part of the basement membrane was exposed or incomplete, exfoliated epithelial cells were seen in the lumen, many small tubules were filled with cell debris, and the lumen was narrowed or blocked , diffuse interstitial edema. There were no obvious pathological changes in the glomeruli (Figure 3).

II. Paller's score for renal tubular injury: 10 high-power fields are selected for each specimen, and 10 renal tubules with the most severe lesions are scored, and a total of 100 renal tubules with lesions are counted. Scoring criteria: renal tubules are significantly dilated , renal tubular epithelial cells flattened 1 point; brush border shedding points; renal tubular epithelial cell vacuolar degeneration 1-2 points; interstitial edema 1 point; lumen obstruction or cast 1-2 points; tubular epithelial cell necrosis 1 -2 points; renal tubular epithelial cell membrane vesicles formed 1-2 points.

The results of Paller's score of renal tubular damage, the control group and the experimental group showed that the renal tubules were damaged during the cryopreservation process, but the score of the experimental group treated by the method of the present invention was lower than that of the cryopreserved control group (Fig. 4).

detect

① Dewaxing and hydration process as described above

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

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

④ Shake off the excess liquid on the slice and add 100µL of TUNEL reaction solution dropwise. The negative control used distilled water instead of TdT enzyme. Incubate at 37°C for 60min in the dark;

⑤ Rinse with PBS for 5min×3, mix the DAPI staining solution with the aqueous mounting medium and mount the slides;

⑥ Observed under microscope, 5 fields of view were randomly taken from each slice to take pictures and the number of apoptotic cells was evaluated.

The morphological changes of nuclear chromatin in the process of apoptosis are divided into three phases: the nuclei in phase I are corrugated or crease-like, and part of the chromatin is condensed; the chromatin in phase IIa nuclei is highly condensed and marginalized; IIb The nuclei in the phase are lysed into fragments, resulting in 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 the organ treated by the method of the present invention was less than that in the control group (Fig. 5).

Claims (2)

1. a method for reducing ice crystal damage of cryopreserved tissues and organs, is characterized in that, 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 min; The mesenchymal stem cell conditioned medium is a mesenchymal stem cell of one of adipose, umbilical cord, umbilical cord blood or bone marrow within 5 generations of passage, which grows to 70-80% confluence after 24 hours of passage, and replaces serum-free mesenchymal stem cells. Stem cell culture medium, the culture supernatant collected after 48 hours is centrifuged at 1000~3000g for 10~30min, and the supernatant is taken and filtered with a 0.1µm~0.22µm microporous membrane; 2) Then perfuse the organ with cryoprotectant pre-cooled to 4°C for 30 minutes; 3) Put the processed organs into freezer bags, seal them, and place them in a program-controlled cooling device. Perform programmed cooling according to the set cooling program. After the temperature drops to -80°C, put them into liquid nitrogen for storage; The program-controlled cooling device cooling program is set as ① 4 ℃ balance, ② drop to 0 ℃ at a speed of 1 ℃/min, and keep for 10 min, ③ drop to -18 ℃ at a speed of 2 ℃/min, keep for 15 min, ④ with a 2°C/min speed to -45°C, hold for 15min, ⑤ Then drop to -90°C at 5°C/min speed, hold for 5min; 4) When resuscitating organs, take out the frozen organs from liquid nitrogen and place them in a 37°C water bath with constant shaking; 5) After complete thawing, perform perfusion again. The perfusion solution is conditioned medium containing mesenchymal stem cells at 12-25°C, and the perfusion time is 30-60 minutes; the treated organs can be directly transplanted. 2 . The method according to claim 1 , wherein the conditioned medium containing mesenchymal stem cells has a cell density of 1×10 ⁷ cells/L and a perfusion rate of 1.5 mL/min. 3 .

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