CN106719599B - Method for reducing ice crystal damage of deep low temperature frozen tissue and organ - Google Patents
Method for reducing ice crystal damage of deep low temperature frozen tissue and organ Download PDFInfo
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- CN106719599B CN106719599B CN201611063329.8A CN201611063329A CN106719599B CN 106719599 B CN106719599 B CN 106719599B CN 201611063329 A CN201611063329 A CN 201611063329A CN 106719599 B CN106719599 B CN 106719599B
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- 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
- A01N1/02—Preservation of living parts
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- 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
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- 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
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0247—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components for perfusion, i.e. for circulating fluid through organs, blood vessels or other living parts
Abstract
The invention provides a method for reducing ice crystal damage of a deep low-temperature cryopreserved tissue organ, which is characterized in that a stem cell conditioned medium is used for perfusion before the organ is frozen, so that the survival state of tissue cells is improved, the anti-apoptosis capacity is increased, and the oxidative stress damage of the cells after in vitro is reduced; after recovery, a complete culture medium containing fresh mesenchymal stem cells is immediately used for post-perfusion treatment, cells which are subjected to toxicity and permeation damage of a cryoprotectant in the cryopreservation process are actively repaired, the apoptosis rate of the cells can be effectively reduced, and the damage of a tissue structure is reduced.
Description
Technical Field
The invention belongs to the field of organ tissue deep low temperature cryopreservation in clinical medicine, and discloses a pretreatment and post-treatment method for reducing apoptosis and damage of ice crystals to cell membranes by using mesenchymal stem cells and secretion products thereof for perfusion before and after organ tissue deep low temperature cryopreservation.
Background
Organ transplantation is the final means for treating severe organ failure, but donor donation and recipient preparation always have time and space difference, so that further development of safer and longer-acting organ cryopreservation-resuscitation method becomes a problem to be solved urgently.
The freezing and freezing process of the biological tissue comprises the steps of filling of a freezing protective agent, program-controlled cooling, deep low-temperature storage, resuscitation and thawing and replacement and cleaning of the freezing protective agent. Each step affects the viability of cells and the maintenance of tissue structure function in the organ after freezing. In 1972, Mazur et al first proposed a two-factor hypothesis of freezing damage from experiments in which cultured Chinese hamster tissue cells were cryopreserved at low temperatures. One is the solute damage effect, which means that the cooling rate is too slow, resulting in an increase in extracellular solute concentration. The other is the mechanical damage effect, which means that the ice crystal formed in the cell directly damages the membrane structure of the cell due to the over-high cooling speed.
In order to reduce damage of freezing to cells, vitrification freezing is mostly used at present, so that the viscosity of liquid is improved, and the formation of internal crystals is reduced in the freezing and curing process. Vitrification of the solution is achieved primarily by increasing the cooling rate and increasing the solution concentration, which requires the use of cryoprotectants. However, the cryoprotectant itself may also damage cells, the high concentration protectant may have toxic effects on cells and tissues, and osmotic pressure difference between the inside and outside of cell membranes may be caused during elution after rewarming, resulting in osmotic damage. Therefore, the focus of today's cryogenic research is on reducing cryoprotectant damage during vitrification. The existing experiment direction mainly improves the introduction and elution modes, and uses a continuous introduction method; a negative pressure impregnation technology for accelerating the penetration of the protective agent; the different kinds of cryoprotectants are used for mutually diluting the cryoprotectants. However, in addition to their properties, cryoprotectants also have different protective and toxic effects in different tissues. The organ is not a simple set of various cells, and is used as a complex tissue with a complex structure, and the difficulty of low-temperature cryopreservation is greatly increased due to the difference of cooling rates caused by uneven temperature distribution of all parts and different sensibility of various cells to cryoprotectants in the cooling process. Therefore, it is very important to find new methods for protecting tissue cells from freezing and repairing damage.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for carrying out tissue pre-perfusion and post-perfusion by applying umbilical cord mesenchymal stem cells and a conditioned medium thereof in the process of freezing and cryopreserving organs, which can effectively improve the self-repairing capability of tissues, effectively improve the proportion of living cells in the resuscitated tissues and enable the tissues and organs to have better recovery function after rewarming.
The invention relates to a deep low temperature cryopreservation method of organs, which comprises the following steps:
1) connecting and pre-cooling an organ to be frozen to a gradient concentration mixed program-controlled cooling organ perfusion instrument at 4 ℃, and perfusing a mesenchymal stem cell conditioned medium with a constant temperature of 25 ℃ for 30-60 min;
the mesenchymal stem cell conditioned medium is mesenchymal stem cells of one of fat, umbilical cord, cord blood or bone marrow in 5 generations of passage, the mesenchymal stem cells grow to 70-80% confluence after 24 hours after passage, a serum-free mesenchymal stem cell culture medium is replaced, culture supernatant collected after 48 hours is centrifuged for 10-30 min at 1000-3000 g, and the culture supernatant is filtered by a microfiltration membrane of 0.1-0.22 mu m;
2) then perfusing the organ with cryoprotectant pre-cooled to 4 deg.C for 30 min;
3) placing the treated organ into a freezing bag, sealing, placing into a program-controlled cooling instrument, performing programmed cooling according to a set cooling program, and storing in liquid nitrogen after the temperature is reduced to-80 deg.C;
the program-controlled cooling program of the cooling instrument is set as 4 ℃ balance, the temperature is reduced to 0 ℃ at the speed of 1 ℃/min and kept for 10min, the temperature is reduced to-18 ℃ at the speed of 2 ℃/min and kept for 15min, the temperature is reduced to-45 ℃ at the speed of 2 ℃/min and kept for 15min, and the temperature is reduced to-90 ℃ at the speed of 5 ℃/min and kept for 5 min;
4) when the organs are recovered, the frozen organs are taken out from liquid nitrogen and then placed in a water bath at 37 ℃ and continuously shaken;
5) pouring again after completely melting, wherein the pouring liquid is a culture medium containing mesenchymal stem cells at the temperature of 12-25 ℃, and the pouring time is 30-60 min;
the treated organ can be directly transplanted.
The mesenchymal stem cell-containing conditioned medium has a cell density of 1x107one/L, perfusion rate 1.5 mL/min.
According to the processing method for cryopreserving the organ at the low temperature, the stem cell conditioned medium is used for perfusion before the organ is frozen, so that the survival state of the tissue cells is improved, the anti-apoptosis capacity is improved, and the oxidative stress damage of the cells after the cells are isolated is reduced; after recovery, a complete culture medium containing fresh mesenchymal stem cells is immediately used for post-perfusion treatment, cells which are subjected to toxicity and permeation damage of a cryoprotectant in the cryopreservation process are actively repaired, the apoptosis rate of the cells can be effectively reduced, and the damage of a tissue structure is reduced.
Drawings
FIG. 1 is a flow chart of the organ cryopreservation method provided by the invention;
FIG. 2 renal artery cannulation and ligation fixation;
FIG. 3 HE staining results of each group of specimens;
a is fresh rat kidney paraffin section HE staining (40 ×);
b is HE staining of fresh rat kidney paraffin sections (200 ×);
c is HE staining (40 x) of paraffin sections of rat kidney of a frozen control group;
d is the HE staining (200 x) of the paraffin section of the rat kidney of the frozen control group;
e is HE staining of MSC perfused rat kidney paraffin sections (40 ×);
f is HE staining of MSC perfused rat kidney paraffin sections (200 ×);
FIG. 4. renal tubule Paller score of rats in each experimental group;
FIG. 5. TUNEL staining results for each group of specimens;
fresh rat kidney paraffin section TUNEL staining (200 ×);
c, d. cryopreserving control rat kidney paraffin sections TUNEL staining (200 ×);
e, f, MSC perfused group rats kidney paraffin sections TUNEL stained (200 ×).
Detailed Description
In order to clarify the objects, technical solutions and advantages of the present invention, the following is a clear and complete description of the technical solutions of the present invention, taking rat kidney as an example.
Example 1
Cryopreservation and resuscitation of rat kidney
And (4) taking the adipose-derived mesenchymal stem cells cultured to the 4 th-6 th generation, changing a fresh culture medium after passage, and collecting culture supernatant for later use after 48 hours.
SD rats of 8-12 weeks old are selected and are anesthetized by intraperitoneal injection of chloral hydrate. The abdominal cavity was opened and the kidneys were removed, taking care to keep the renal artery and vein intact.
The kidney was washed in 4 ℃ physiological saline and placed in the same precooled stem cell conditioned medium.
Fixing with parallel silk thread by renal artery intubation, ligating renal artery branch, connecting with program-controlled cooling organ perfusion apparatus precooled to 4 deg.C, starting quantitative liquid inlet pump, perfusing stem cell conditioned medium to kidney at 1.5mL/min for 60min (fig. 2).
The perfusate was changed to cryoprotectant and perfusion was performed in the same manner.
Loading the filled kidney into a freezing bag, placing the kidney in a programmed cooling instrument, setting the program as (1) 4 ℃ balance, (0) 10min for cooling at the speed of 1 ℃/min, (18) 15min for cooling at the speed of 2 ℃/min, (45) 15min for cooling at the speed of 2 ℃/min, (90) 5min for cooling at the speed of 5 ℃/min. The cryopreservation bag was then put into liquid nitrogen.
After one week of freezing, the frozen kidney was taken out, placed in a 37 ℃ water bath to melt, and shaken constantly to make the temperature distribution uniform.
Digesting the adipose-derived mesenchymal stem cells cultured to the 4 th-6 th generation by using pancreatin, and re-suspending and adjusting the cell concentration to be 1 × 10 by using a conditioned medium pre-cooled to 4 DEG C7And (2) per liter.
After the organ was completely thawed in the water bath, the organ was transferred to the stem cell conditioned medium, the renal artery cannula was again connected to the perfusion apparatus, and perfusion was performed using the stem cell suspension described in step 5, at a rate of 1.5mL/min for 30 min.
Example 2
Determination of Activity after Resuscitation of cryopreserved organs
Rat kidneys were divided into three groups in the cryopreservation experiment, respectively:
fresh group: rat kidneys were taken and immediately fixed in 4% paraformaldehyde without cryopreservation.
Control group: the frozen stock is carried out according to the common frozen stock method without stem cell treatment.
Experimental groups: the cryopreservation is carried out according to the method disclosed by the invention.
The organs of each group were thawed one week after cryopreservation and their structural integrity and cellular activity were determined.
Morphological analysis
Preparation of Paraffin section of tissue
Paraffin embedding and slicing: each group of kidneys was coronal dissected through the renal arteries, and half of the tissues were fixed in 4% paraformaldehyde. The other half of the tissue was used for molecular biology testing. Fixed tissues are dehydrated, transparent, waxed and embedded to prepare paraffin tissue blocks, and then coronal continuous slicing is carried out on a paraffin slicer, wherein the thickness of the fixed tissues is 4 um.
Hematoxylin-eosin staining (HE staining)
Dewaxing and hydrating: slicing paraffin, baking at 60 deg.C for 30min, sequentially adding xylene 20min × 2, anhydrous ethanol 10min × 2,95% ethanol 5min × 2,90% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min, and PBS 5min × 3.
Dyeing and differentiating: the sections were stained for 10min by immersing in hematoxylin stain, 1% hydrochloric acid alcoholic solution was dropped for 5s, and the reverse blue was rinsed with tap water for 10 min.
Counterdyeing: soaking in 0.5% eosin dye solution for 2-5min, and washing with PBS for 10 min.
Dehydrating and sealing with conventional gradient ethanol, soaking the stained section in 80% ethanol for 5min, 90% ethanol for 5min, 95% ethanol for 10min × 2, anhydrous ethanol for 10min × 2, xylene for 10min × 2, and sealing with neutral gum.
And (5) observing and taking a picture under a light mirror.
And (3) morphological analysis:
I. after cryopreservation and resuscitation, the kidney of the control and MSC perfusion groups can see the pathological changes with the main characteristics of the acute degeneration and necrosis of the proximal tubule in the renal cortex region; the brush border of the renal tubular epithelial cells is shed, the cells are flattened, the tubular cavity of the renal tubular is expanded, partial cell vacuole degeneration and swelling rupture, nucleus concentration, rupture and even disappearance are realized, partial basement membrane is naked or incomplete, the shed epithelial cells can be seen in the tubular cavity, the tubular cavity of a plurality of positions is filled with cell debris, the tubular cavity is narrowed or blocked, and interstitial edema is diffused. Glomeruli were not significantly pathologically changed (fig. 3).
Paller's score for renal tubular injury: for each specimen, 10 high power field views were selected and the 10 most diseased tubules were scored for a total of 100 diseased tubules, with scoring criteria: clear dilation of renal tubules, flattened 1 minute of renal tubular epithelial cells; the brush edge falls off; vacuolar degeneration of renal tubular epithelial cells for 1-2 points; interstitial edema 1 point; obstruction of lumen or tube type 1-2 points; necrosis of renal tubular epithelial cells is divided into 1-2 points; the membrane vesicle of the renal tubular epithelial cell forms 1 to 2 points.
The results of the Paller's score for renal tubular injury, the control and experimental groups showed renal tubular injury during cryopreservation, but the experimental group treated by the method of the present invention scored less than the cryopreservation control group (FIG. 4).
Detection of
Dewaxing hydration process is as described above
② immersing the slices into 3 percent hydrogen peroxide solution to incubate for 10min at room temperature. Washing with PBS for 2min × 3;
thirdly, immersing the slices into 100mM Tris/HCl containing 10 mug/L protease K, digesting for 20min at 37 ℃, and washing for 5min multiplied by 3 by PBS;
and fourthly, throwing off redundant liquid on the slices, and dropwise adding 100 mu L of TUNEL reaction solution. The negative control used distilled water instead of TdT enzyme. Incubating at 37 deg.C in dark for 60 min;
washing with PBS for 5min × 3, mixing DAPI staining solution with water-based sealing agent, and sealing;
sixthly, observing under a lens, randomly taking 5 fields for each section, photographing and evaluating the number of the apoptotic cells.
Morphological changes in nuclear chromatin during apoptosis are divided into three phases: the nucleus in stage I is wavy or creased, and part of chromatin is in condensed state; chromatin of IIa nuclei is highly condensed and marginalized; the nuclei of phase IIb are cleaved into fragments, producing apoptotic bodies. Apoptotic cells were visualized green under a laser microscope after TUNEL staining.
The results show that the organ treated by the method of the invention has fewer apoptotic cells than the control group (FIG. 5).
Claims (2)
1. A method for reducing ice crystal injury of deep low temperature frozen tissue organs is characterized by comprising the following steps:
1) connecting and pre-cooling an organ to be frozen to a gradient concentration mixed program-controlled cooling organ perfusion instrument at 4 ℃, and perfusing a mesenchymal stem cell conditioned medium with a constant temperature of 25 ℃ for 30-60 min;
the mesenchymal stem cell conditioned medium is mesenchymal stem cells of one of fat, umbilical cord, cord blood or bone marrow in 5 generations of passage, the mesenchymal stem cells grow to 70-80% confluence after 24 hours after passage, a serum-free mesenchymal stem cell culture medium is replaced, culture supernatant collected after 48 hours is centrifuged for 10-30 min at 1000-3000 g, and the culture supernatant is filtered by a microfiltration membrane of 0.1-0.22 mu m;
2) then perfusing the organ with cryoprotectant pre-cooled to 4 deg.C for 30 min;
3) placing the treated organ into a freezing bag, sealing, placing into a program-controlled cooling instrument, performing programmed cooling according to a set cooling program, and storing in liquid nitrogen after the temperature is reduced to-80 deg.C;
the program-controlled cooling program of the cooling instrument is set as 4 ℃ balance, the temperature is reduced to 0 ℃ at the speed of 1 ℃/min and kept for 10min, the temperature is reduced to-18 ℃ at the speed of 2 ℃/min and kept for 15min, the temperature is reduced to-45 ℃ at the speed of 2 ℃/min and kept for 15min, and the temperature is reduced to-90 ℃ at the speed of 5 ℃/min and kept for 5 min;
4) when the organs are recovered, the frozen organs are taken out from liquid nitrogen and then placed in a water bath at 37 ℃ and continuously shaken;
5) pouring again after completely melting, wherein the pouring liquid is a culture medium containing mesenchymal stem cells at the temperature of 12-25 ℃, and the pouring time is 30-60 min; the treated organ can be directly transplanted.
2. The method of claim 1, wherein the mesenchymal stem cell-containing conditioned medium has a cell density of 1x107one/L, perfusion rate 1.5 mL/min.
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| CN103210903A (en) * | 2013-05-03 | 2013-07-24 | 新乡医学院 | Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof |
| CN104938477A (en) * | 2015-04-10 | 2015-09-30 | 杭州阿德莱诺泰制药技术有限公司 | CIK (cytokine-induced killer) frozen stock solution and frozen preservation method |
| CN105831106A (en) * | 2016-05-10 | 2016-08-10 | 天津普瑞赛尔生物科技有限公司 | Cryopreservation method of DC cells and CIK seed cells in blood, prepared cells and application |
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| WO2014193895A1 (en) * | 2013-05-29 | 2014-12-04 | Allocure, Inc. | Ex vivo perfusion of donor organs prior to transplantation using mesenchymal stem cells |
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| CN103210903A (en) * | 2013-05-03 | 2013-07-24 | 新乡医学院 | Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof |
| CN104938477A (en) * | 2015-04-10 | 2015-09-30 | 杭州阿德莱诺泰制药技术有限公司 | CIK (cytokine-induced killer) frozen stock solution and frozen preservation method |
| CN105831106A (en) * | 2016-05-10 | 2016-08-10 | 天津普瑞赛尔生物科技有限公司 | Cryopreservation method of DC cells and CIK seed cells in blood, prepared cells and application |
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