CN110317782B - Method for improving survival of mesenchymal stem cells after resuscitation and frozen stock solution used by method - Google Patents

Method for improving survival of mesenchymal stem cells after resuscitation and frozen stock solution used by method Download PDF

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CN110317782B
CN110317782B CN201910652401.8A CN201910652401A CN110317782B CN 110317782 B CN110317782 B CN 110317782B CN 201910652401 A CN201910652401 A CN 201910652401A CN 110317782 B CN110317782 B CN 110317782B
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cells
cell
mesenchymal stem
umbilical cord
culture
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CN110317782A (en
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王小武
迟大明
朱春颖
郭春明
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Tianjin Ruibosi Biotechnology Co ltd
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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
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a method for improving survival of mesenchymal stem cells after resuscitation and a freezing solution used by the method. In particular, in one aspect, the present invention relates to a method for cryopreserving and resuscitating mesenchymal stem cells, the method comprising the steps of: uniformly mixing the prepared mesenchymal stem cells with cell freezing solution, and then freezing the cell solution in liquid nitrogen until the cells are used; when the cells need to be revived, the frozen cells are taken out from the liquid nitrogen and rapidly put into a water bath at 37 ℃ to completely melt the frozen solution, and the cells are transferred to an ice bath at 4 ℃ after being melted, so that the cell reviving is completed. Another aspect relates to a cell cryopreservation solution for cryopreservation and resuscitation of mesenchymal stem cells, comprising DMEM-F12, dimethyl sulfoxide, canine blood albumin, and the like. Also relates to a method for preparing mesenchymal stem cells from the umbilical cord of a dog and for cryopreservation. The method and the cell freezing solution of the invention have excellent technical effects as described in the specification.

Description

Method for improving survival of mesenchymal stem cells after resuscitation and frozen stock solution used by method
Technical Field
The present invention is in the field of stem cell therapy, for example, stem cell therapy of canine diseases, to a method of preparing mesenchymal stem cells (from, for example, the umbilical cord of a canine), and to the use of such mesenchymal stem cells in the treatment of, for example, canine diseases. The method for preparing the mesenchymal stem cells from the umbilical cord of the dog has excellent technical effects. The obtained mesenchymal stem cells can be used for treating canine arthritis, fracture, muscle injury, ligament injury, cartilage injury, joint injury, cognitive dysfunction, immune-mediated diseases, xerophthalmia, recurrent uveitis, liver diseases, heart diseases, kidney diseases, diabetes, gastrointestinal diseases, thyroid diseases and skin diseases. Furthermore, the invention also relates to a method for cryopreserving the mesenchymal stem cells prepared from the umbilical cord of the dog, and in particular, the related cells can maintain the cell viability for a longer time after being thawed. In addition, the mesenchymal stem cells of the present invention may also be derived from cats; in addition, the mesenchymal stem cells of the present invention may be a canine or feline derived fat or umbilical cord.
Background
Mesenchymal stem cells (mesenchymal stem cells, MSCs or MSCs) are adult stem cells present in the interstitial tissue of the body and can be obtained from a variety of sources such as placenta, umbilical cord, bone marrow, adipose tissue, etc. MSCs have a strong self-proliferative capacity and a multidirectional differentiation potential. Under controlled conditions, it can differentiate into various cell types such as nerve cells, cartilage cells, fat cells, cardiac muscle cells and osteoblasts in vivo or in vitro. At the same time, MSC has immune compatibility, does not cause immune rejection reaction, and can be used for allogeneic transplantation. In addition, MSC has no tumorigenicity, but can support hematopoiesis and secrete a plurality of beneficial cytokines and immune factors, so that the MSC can be safely used for tissue regeneration and repair treatment, and has huge treatment potential in the aspects of the difficult and complicated symptoms of the prior medical means. In the veterinary field, stem cell therapies are effective in improving the quality of life of animals and helping them get rid of the affliction of illness. The dogs are important companion animals, can be trained to work dogs such as search dogs, guide dogs and the like, and are also important animal models for new drug evaluation and preclinical drug experiments. Currently, in the field of canine stem cell therapy, there have been many cases of successful treatment of canine arthritis, fractures, muscle, ligament or cartilage/joint injuries by mesenchymal stem cell injection. Other treatments such as canine cognitive dysfunction, immune-mediated diseases, dry eye, recurrent uveitis, various liver diseases, heart diseases, kidney diseases, diabetes, gastrointestinal diseases, thyroid problems, skin diseases, and the like are also being evaluated.
The great application prospect is urgent to establish a high-efficiency canine mesenchymal stem cell separation preparation method so as to separate high-quality MSC in large scale. The MSC sources commonly used on dogs are fat and bone marrow, but the process of obtaining stem cells from these two sources inevitably causes trauma and pain to the donor animal, and although placenta/umbilical cord are waste from animal production, they are also one of the rich sources of MSCs, so that animals can be better protected by their isolation of cells as samples.
CN108456657a (chinese patent application No. 201810278145.6) discloses a method for preparing mesenchymal stem cells from the umbilical cord of dogs and/or a cryopreservation method comprising the steps of: (1) disinfection and cleaning: sterilizing the surface of the umbilical cord tissue of the dog by using a sterilizing liquid, shearing and spreading the umbilical cord, and cleaning the umbilical cord tissue by using a buffer solution to reduce red blood cells on the umbilical cord tissue; (2) digestion treatment: cutting umbilical cord tissue obtained in the step (1) into tissue blocks, putting the tissue blocks into a digestive enzyme solution, performing digestion treatment for 0.5-3 hours, filtering to remove the tissue blocks, adding a mesenchymal stem cell culture medium to terminate digestion, and then performing cell cleaning on cells obtained by digestion to finally obtain a cell suspension; (3) cell culture: placing the cell suspension obtained in the step (2) into a culture container, placing the culture container into an incubator for culture, taking the culture container out of the incubator when the culture is carried out for 2-7 days, supplementing a proper amount of mesenchymal stem cell culture medium, and continuing to culture; taking the culture container out of the incubator at 8-11 days, performing the first full liquid exchange, and continuing culturing; performing full liquid change every 1-3 days; (4) passage of cells: separating the adherent cells from the bottom of the culture container by using digestive enzyme after the fusion rate of the adherent cells in the culture container reaches 40% -70%, centrifuging, pumping out supernatant, adding a mesenchymal stem cell culture medium to re-suspend the cells, inoculating the cells into the culture container for culture, and changing the liquid once every 1-3 days until the fusion rate reaches 70% -90%, thereby obtaining the P1-generation umbilical cord mesenchymal stem cells; then carrying out necessary passage according to the culture method; optionally (5) cryopreserving: and (3) adding the cell freezing solution into the umbilical cord mesenchymal stem cells obtained in the step (4), and freezing in liquid nitrogen for later use. Specifically, in this CN108456657a an improved cell cryopreservation solution is disclosed comprising the following components: the modified cell cryopreservation liquid is used for cryopreserving the P1 to P15 generation cells, namely, after the cryopreservation-resuscitation process, the survival percentage of the cells is 92-96%, which shows that the cell death caused in the cell cryopreservation-resuscitation process can be remarkably reduced when a trace amount of dextran is added into the cryopreservation liquid.
Cell resuscitation is the opposite process to cell cryopreservation, i.e., the process of cell growth recovery, in which cells frozen in liquid nitrogen (-196 ℃) or in a refrigerator (dry ice, -60 ℃ to-80 ℃) are thawed and then re-cultured or used. When the cell is restored to the normal temperature state, the morphological structure of the cell is kept normal, and the biochemical reaction can be restored. Unlike cell cryopreservation, the temperature rise in the cell resuscitation process is rapid, preventing moisture from entering the cells during thawing, forming ice crystals, and affecting cell survival. In cell resuscitation, a freezing pipe is taken out of liquid nitrogen, rapidly put into a water bath at 37 ℃ to enable frozen stock solution to be completely melted within 1-5 min, and after the cells are melted, the frozen stock solution is removed from the water bath at 37 ℃ as soon as possible and placed on an ice bath at 4 ℃ to finish cell resuscitation; the resuscitated cells may then be subjected to: detection (e.g., detection of items such as cell count, viability, etc., e.g., viability of resuscitated cells can be detected by trypan blue staining), culturing and/or expansion (e.g., passaging), and/or biological applications (e.g., animal testing or clinical stem cell therapy), etc. However, in practical applications of stem cells, the cells usually need to be left for a period of time varying from several hours to several tens of hours in order to perform other necessary procedures prior to use, which is sometimes at a temperature of 25 ℃, usually at a temperature of 4 ℃. Unfortunately, it has been found that stem cells present in existing cryopreservation solutions exhibit a problem of insufficient stability during the above-mentioned storage for several to several tens of hours at 25 ℃ or 4 ℃, which is manifested in that the cell viability, i.e. the survival percentage, is significantly reduced with prolonged storage time.
Thus, there is an urgent need in the art for new methods to improve the stability of post-resuscitated stem cells prior to use.
Disclosure of Invention
It is another object of the present invention to provide a method for preparing mesenchymal stem cells from umbilical cord of canine, which is to provide a method for improving stability of stem cells after resuscitating before use, especially because the cells after resuscitating usually need to be left for several hours to tens of hours in order to perform other necessary procedures before use, which is occasionally carried out at 25 ℃ temperature, usually at 4 ℃ temperature, and the stem cells present in some cryopreservation solutions may exhibit a problem of insufficient stability during the period of several hours to tens of hours at 25 ℃ or 4 ℃ as described above, which is manifested in that cell viability, i.e., survival percentage, may be significantly reduced with prolonged leaving time. It has been unexpectedly found that the preparation of mesenchymal stem cells from the umbilical cord of a canine using the method of the present invention exhibits encouraging effects, and the present invention has been completed based on this finding. In addition, the mesenchymal stem cells of the present invention may also be derived from cats; in addition, the mesenchymal stem cells of the present invention may be a canine or feline derived fat or umbilical cord.
To this end, a first aspect of the present invention provides a method of preparing mesenchymal stem cells from the umbilical cord of a canine and/or a cryopreservation method, the method comprising the steps of:
(1) Sterilizing and cleaning: disinfecting the surface of the umbilical cord tissue (e.g., fresh ex vivo tissue) of the dog with a disinfecting solution (e.g., alcohol, e.g., 75% ethanol as the disinfecting solution), shearing the umbilical cord open (e.g., plated in a culture dish having a diameter of 5-20cm, e.g., a 10cm culture dish), and washing the umbilical cord tissue with a buffer (e.g., PBS buffer, e.g., 0.025M sodium dihydrogen phosphate buffer, ph 6.5) to reduce red blood cells on the umbilical cord tissue; (this step is typically performed in a biosafety cabinet)
(2) Digestion treatment: cutting umbilical cord tissue (in another cell culture dish) obtained in step (1) into tissue blocks (for example, tissue blocks with a size of about 0.05-0.5 cubic centimeters, preferably about 0.05-0.2 cubic centimeters, preferably about 0.1 cubic centimeters), placing the tissue blocks into a digestive enzyme solution (for example, containing type I collagenase and DMEM-F12, and preparing the tissue blocks by, for example, adding 0.1g of type I collagenase into 100ml of DMEM-F12, and then filtering the tissue blocks with a 0.45 μm filter to obtain a digestive solution), performing digestion for 0.5-3 hours (for example, digestion for 1-2 hours at 37 ℃ for 1.5 hours), filtering and removing the tissue blocks (for example, by a filter screen with a size of 50-150 μm, preferably about 100 μm), adding a mesenchymal stem cell culture medium to terminate the digestion, and then performing cell washing on the digested cells to obtain a cell suspension; (As not otherwise described, the mesenchymal stem cell medium used in the present invention contains 15 parts by weight of FBS, 1 part by weight of L-Glutamine, 0.05 part by weight of Gentamicin and 84 parts by weight of DMEM-F12)
(3) Cell culture: placing the cell suspension obtained in step (2) into a culture vessel (e.g., at a density of 0.2-2X 10 4 /cm 2 Added to the culture vessel, preferably at a density of about 1X 10 4 /cm 2 Adding), placing the culture container into an incubator for culture until the culture container is taken out from the incubator until the culture is carried out for 2-7 days (for example, 3-6 days, for example, 4 days, for example, 5 days), supplementing a proper amount (for example, 3 ml) of mesenchymal stem cell culture medium, and continuing the culture; taking the culture container out of the incubator at 8-11 days (such as 9 days), performing the first full liquid exchange, and continuing culturing; performing total liquid exchange every 1-3 days (e.g. 2 days) later;
(4) Cell passage: when the adherent cell fusion rate in the culture vessel reaches 40% -70% (e.g., 60%), a digestive enzyme is used (e.g., in the present invention, trypLE is used, as not specifically described TM Express) separating the adherent cells from the bottom of the container, centrifuging, pumping out supernatant, adding a mesenchymal stem cell culture medium to re-suspend the cells, inoculating the cells into the culture container for culture, and changing the liquid once every 1-3 days (for example every 2 days) until the fusion rate reaches 70-90% (for example 80%), thus obtaining the P1-generation umbilical cord mesenchymal stem cells; next, necessary passages (e.g., trypLE used in the present invention) are performed according to the above-described culture method TM Express, its composition is: 200.0mg/L of potassium chloride, 200.0mg/L of potassium dihydrogen phosphate, 8000.0mg/L of sodium chloride, 2160.0mg/L, EDTA 457.6mg/L of disodium hydrogen phosphate heptahydrate and commercial amounts of rProtease; the TrypLE TM Express is commercially available from the company Simer Feier, see for example http:// www.thermofisher.com/cn/zh/home/technical-resources/media-formulation.346.Html for information on its technology; optionally (I)
(5) Freezing: and (3) adding cell freezing solution (for example, adding the cell freezing solution in the volume ratio of 1:1) into the umbilical mesenchymal stem cells obtained in the step (4) and freezing the umbilical mesenchymal stem cells in liquid nitrogen for later use. (e.g., the cell cryopreservation solution comprises DMEM-F12, dimethyl sulfoxide and canine blood albumin. In one embodiment, the cell cryopreservation solution comprises about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide, about 15 parts of canine blood albumin.
According to the method of the first aspect of the invention, the PBS buffer in step (1) is formulated as sodium and/or potassium salts of phosphoric acid having a pH of 5.0-8.0, preferably a pH of 5.5-76, preferably a pH of 6.0-7.0. In one embodiment, the phosphate concentration in the PBS buffer is 0.01 to 0.5M, preferably 0.02 to 0.1M. In the experiments described below according to the invention, the PBS buffer used was sodium phosphate, where the phosphate concentration was 0.025M and the pH was 6.5, as not specified. The inventors found that the concentration of the PBS buffer and the pH value in the above-described range had little effect on the effect of the method of the present invention.
According to the method of the first aspect of the present invention, the digestive enzyme solution in the step (2) is obtained by adding collagenase type I to DMEM-F12, filtering the mixture through a filter, wherein the digestive enzyme is 0.05g to 0.5g, preferably 0.08g to 0.2g, preferably 0.1g, the digestive enzyme is 50 to 500ml, preferably 80 to 200ml, preferably 100ml, of DMEM-F12, and the filter is 0.45 μm. In one embodiment, the digestive enzyme solution is obtained by adding 0.1g type I collagenase to 100ml DMEM-F12, mixing well, and filtering (e.g., with a 0.45um filter).
The method according to the first aspect of the invention, wherein in step (2) the tissue mass has a cubic block shape with a size of about 0.05 to about 0.5 cubic centimeter, preferably about 0.05 to about 0.2 cubic centimeter, preferably about 0.1 cubic centimeter.
The method according to the first aspect of the invention, wherein in step (2) the tissue mass has a size of 0.05-0.5 cubic centimeter, preferably 0.05-0.2 cubic centimeter, especially a size of about 0.1 cubic centimeter. Although small tissue fragments are expected to be advantageous for the practice of the method of the invention, the inventors have found in experiments that in the three conditions of 0.05 cubic cm, 0.1 cubic cm and 0.5 cubic cm they are substantially identical to the digestion treatment effect of the digestive enzyme, whereas a volume of greater than 1 cubic cm has a significant adverse effect on the digestion effect of the digestive enzyme, which may be somewhat weakened by extending the digestion time.
The method according to the first aspect of the present invention, wherein in step (2), the digestion treatment is carried out for a period of 0.5 to 3 hours, preferably 1 to 2.5 hours, preferably 1.5 to 2 hours. The inventor finds that the digestion treatment effect on the tissue block is optimal within the digestion treatment time of 1-2.5 hours, so that the tissue block can be ensured to be fully digested, and cells can be prevented from being damaged.
The method according to the first aspect of the invention, wherein in step (2), the digestion treatment is carried out at a temperature in the vicinity of the body temperature of the human body, preferably 34-40 ℃, preferably 36-38 ℃, preferably 37 ℃.
The method according to the first aspect of the invention, wherein in step (2), the digestion treatment is carried out in a thermostatted shaker.
The method according to the first aspect of the invention, wherein in step (2) the filtering of the tissue mass is performed by means of a sieve, said sieve being a 50-150 μm sieve, preferably an about 100 μm sieve.
The method according to the first aspect of the present invention, wherein in step (2), the mesenchymal stem cell medium for terminating the digestion is added in a ratio of 2:1 to 1:2, preferably in a ratio of 1:1, said ratio being a volume ratio.
The method according to the first aspect of the present invention, wherein in step (2), the specific step of cell washing is centrifugation for 5-15 minutes, supernatant is removed, PBS buffer is added to resuspend cells, centrifugation is performed for 5-15 minutes again, supernatant is removed, mesenchymal stem cell medium is added, and a small sample is withdrawn for cell counting. The centrifugation speed is 800-2000rpm, preferably 1250rpm, and the centrifugation time is preferably 10 minutes.
The method according to the first aspect of the present invention, wherein the mesenchymal stem cell medium comprises FBS, L-Glutamine (L-glutamic acid), gentamicin (Gentamicin) and DMEM-F12. In one embodiment, the mesenchymal stem cell medium contains 10-20% FBS. In one embodiment, the mesenchymal stem cell medium contains about 15% FBS. In one embodiment, the mesenchymal stem cell medium contains 0.5-2% L-Glutamine. In one embodiment, the mesenchymal stem cell medium contains about 1% L-Glutamine. In one embodiment, the mesenchymal stem cell medium contains about 0.01-0.1% Gentamicin. In one embodiment, the mesenchymal stem cell medium contains about 0.05% Gentamicin. In one embodiment, the mesenchymal stem cell medium contains 80-90% DMEM-F12. In one embodiment, the mesenchymal stem cell medium contains about 84% DMEM-F12. In one embodiment, the mesenchymal stem cell medium contains about 15 parts by weight of FBS, about 1 part by weight of L-Glutamine, about 0.05 part by weight of Gentamicin and about 84 parts by weight of DMEM-F12.
The method according to the first aspect of the invention, wherein the mesenchymal stem cell medium is further supplemented with glycine. For example, the mesenchymal stem cell medium contains 15 parts by weight of FBS, 1 part by weight of L-Glutamine, 0.05 part by weight of Gentamicin, 84 parts by weight of DMEM-F12, and 0.025% w/v glycine.
The method according to the first aspect of the invention, wherein the TrypLE TM 1, 2-propanediol is additionally added to the Express digestive enzyme. For example, the TrypLE TM 0.05% w/v 1, 2-propanediol was additionally added to the Express digestive enzyme. It has been unexpectedly found that supplementation of glycine in the mesenchymal stem cell medium and propylene glycol in the digestive enzymes helps to increase the yield of mesenchymal stem cells.
The method according to the first aspect of the invention, wherein dextran-40 is further added to the frozen stock solution, in one embodiment the frozen stock solution further comprises 0.2-0.5% w/v dextran-40, preferably further comprises 0.25% w/v dextran-40. In one embodiment, the cell cryopreservation solution comprises about 65 parts DMEM-F12, about 10 parts dimethyl sulfoxide, about 15 parts canine blood albumin, 0.2 to 0.5% w/v, particularly 0.25% w/v dextran-40. It has been unexpectedly found that the addition of a minor amount of dextran-40 to the cryopreserved solution helps to increase the survival rate of the cryopreserved cells when revived.
The method according to the first aspect of the present invention, wherein CO in the incubator of step (3) 2 The concentration is 3-7%, preferably 5%, and the temperature of the incubator is controlled within the range of about human body temperature, preferably 34-40deg.C, preferably 36-38deg.C, preferably 37deg.C. Such CO 2 The concentration and temperature are also generally culture conditions commonly used in the art.
Furthermore, in a first aspect of the invention, a method of isolating and expanding mesenchymal stem cells from fresh tissue of canine umbilical cord is provided. Accordingly, in a second aspect the invention provides a canine umbilical cord mesenchymal stem cell.
The canine umbilical cord mesenchymal stem cells according to the second aspect of the present invention, which are obtained according to the method of any one of the embodiments of the first aspect of the present invention.
The canine umbilical cord mesenchymal stem cells according to the second aspect of the present invention have a cell purity of greater than 85%, for example greater than 90%. In one embodiment, the umbilical cord mesenchymal stem cells have a cell purity of greater than 85%, such as greater than 90%, after more than 3 passages.
Further, the third aspect of the present invention relates to the use of the canine umbilical cord mesenchymal stem cells produced according to the present invention in the preparation of a formulation for the treatment and/or prevention of a canine disease selected from the group consisting of: arthritis, bone fracture, muscle injury, ligament injury, cartilage injury, joint injury, cognitive dysfunction, immune-mediated diseases, dry eye, recurrent uveitis, liver diseases, heart diseases, kidney diseases, diabetes, gastrointestinal diseases, thyroid diseases, and skin diseases.
Further, a fourth aspect of the invention provides a method of cryopreserving and resuscitating mesenchymal stem cells (e.g. prepared by a method of any one of the first aspects of the invention), the method comprising the steps of:
(51) Uniformly mixing mesenchymal stem cells (prepared by digestion treatment, cell culture and cell passage processes, such as steps (1) to (4) in the method according to any one of the first aspects of the invention) with a cell freezing solution, and then freezing the cell solution in liquid nitrogen until the cells are used;
(52) When the cells need to be revived, the frozen cells are taken out from the liquid nitrogen and rapidly put into a water bath at 37 ℃ to completely melt the frozen solution within 1-5 min, and the cells are transferred to an ice bath at 4 ℃ after being melted, so that the cell reviving is completed.
According to the method of the fourth aspect of the invention, the volume ratio of the mesenchymal stem cells to the cell cryopreservation solution is 1: 1.
The method according to the fourth aspect of the invention, wherein the cell cryopreservation solution comprises 65 parts DMEM-F12, 10 parts dimethyl sulfoxide, 15 parts canine blood albumin, 0.2 to 0.5% w/v, in particular 0.25% w/v dextran-40.
The method according to the fourth aspect of the invention, wherein the cell cryopreservation solution comprises 65 parts DMEM-F12, 10 parts dimethyl sulfoxide, 15 parts canine blood albumin, 0.06 to 0.08% w/v, e.g. 0.07% w/v povidone K30 and 0.5 to 0.7% w/v, e.g. 0.6% w/v histidine. It has been unexpectedly found that when povidone and histidine are used in combination, not only the percent survival of cells during freeze-recovery is effectively maintained, but also the high survival rate, i.e. excellent survival stability, is maintained for a longer period of time without changing the fluid after cell recovery.
The method according to the fourth aspect of the invention, wherein the cell cryopreservation solution is formulated as follows: uniformly mixing DMEM-F12, dimethyl sulfoxide and canine blood albumin in a specified proportion by volume, and adding povidone K30 and histidine in a specified proportion to dissolve the components to obtain the finished product. The preparation method is a preparation method conventional in the art, and when the frozen stock solution is involved in the invention, the preparation method is similar to the preparation method unless otherwise specified.
The method according to the fourth aspect of the present invention, wherein the mesenchymal stem cells are canine umbilical cord-derived mesenchymal stem cells.
Further, a fifth aspect of the invention provides a cell cryopreservation solution for cryopreservation and resuscitating of mesenchymal stem cells (e.g. prepared by a method according to any one of the first aspects of the invention) comprising 65 parts DMEM-F12, 10 parts dimethyl sulfoxide, 15 parts canine blood albumin, 0.03 to 0.05% w/v, e.g. 0.04% w/v povidone K30 and 0.5 to 0.7% w/v, e.g. 0.6% w/v histidine.
The cell cryopreservation solution according to the fifth aspect of the present invention is formulated as follows: uniformly mixing DMEM-F12, dimethyl sulfoxide and canine blood albumin in a specified proportion by volume, and adding povidone K30 and histidine in a specified proportion to dissolve the components to obtain the finished product.
The cell cryopreservation solution according to the fifth aspect of the present invention, wherein the cryopreservation and resuscitation of mesenchymal stem cells comprises the steps of:
(51) Uniformly mixing mesenchymal stem cells (prepared by digestion treatment, cell culture and cell passage processes, such as steps (1) to (4) in the method according to any one of the first aspects of the invention) with a cell freezing solution, and then freezing the cell solution in liquid nitrogen until the cells are used;
(52) When the cells need to be revived, the frozen cells are taken out from the liquid nitrogen and rapidly put into a water bath at 37 ℃ to completely melt the frozen solution within 1-5 min, and the cells are transferred to an ice bath at 4 ℃ after being melted, so that the cell reviving is completed.
According to the cell freezing solution in the fifth aspect of the invention, the volume ratio of the mesenchymal stem cells to the cell freezing solution is 1: 1.
According to any aspect of the invention, wherein the mesenchymal stem cells may also be cat-derived.
According to any aspect of the invention, wherein the mesenchymal stem cells may be a canine or feline derived fat or umbilical cord.
According to any aspect of the invention, wherein the mesenchymal stem cells are derived from the fat or umbilical cord of a cat, the canine blood albumin used is replaced with feline blood albumin.
The present invention is further described below. The documents cited in the present invention, and the documents cited in the documents, are incorporated by reference in their entirety.
In any of the aspects of the present invention, any technical features thereof are equally applicable to any of the embodiments of any of the aspects of the present invention as long as they do not cause contradiction, and such mutual applicability may be appropriately modified as necessary.
In the present invention, the term "umbilical cord mesenchymal stem cells" refers to mesenchymal stem cells derived from umbilical cord. Thus, in the context of the present invention, and in particular in relation to the present invention, the term "umbilical cord mesenchymal stem cells" may be used interchangeably with "umbilical cord stem cells", "mesenchymal stem cells", unless explicitly indicated otherwise.
In the present invention, the term "PBS buffer" or "PBS" refers to phosphate buffer. The general formulation and method of formulation of PBS for use in the context of the present invention and their general properties such as pH or pH range are well known to those skilled in the art.
In the present invention, the term "umbilical cord" refers to the umbilical cord of a canine, and in particular to the umbilical cord within 4 hours after delivery.
Mesenchymal stem cells (mesenchymal stem cell, MSC) such as those of mammals such as humans or dogs were originally isolated from bone marrow, and a class of tissue stem cells derived from mesoderm that have multipotent differentiation potential and self-renewal capacity, and that have the capacity to differentiate into various osteoblasts, chondrocytes, adipocytes, endothelial cells, neural cells, myocytes, hepatocytes, etc., under specific conditions in vivo and in vitro (Caplan ai.msenchl stem cells.j ortho res.1991,9:641-650.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999;284:143-147). Recent studies have shown that mesenchymal stem cells have immunoregulatory and hematopoietic support effects and are easy for exogenous gene expression. Therefore, the mesenchymal stem cells not only tissue engineer seed cells in bone, cartilage and myocardial construction and carrier cells important in gene therapy, but also have wide application prospects in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft versus host reaction functions. Mesenchymal stem cells have the characteristic of in vitro adherent growth, and by utilizing the characteristic, people have successfully isolated and cultured the mesenchymal stem cells from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like. As is generally understood in the art, in the present invention, as not otherwise specified, the canine blood albumin used is a 10% concentration of canine blood albumin. In the present invention, DMEM-F12 used is readily available from various brands of commercial sources, and in the present invention, gibco brand DMEM-F12 is used, unless otherwise specified. In the present invention, when reference is made to "freezing" it is intended to refer to freezing in liquid nitrogen, unless otherwise specified, and this freezing operation is a conventional operation in the art, and specific operation procedures are as follows: uniformly mixing the prepared mesenchymal stem cells with cell freezing solution, and then freezing the cell solution in liquid nitrogen until the cells are used;
In the present invention, when referring to "resuscitation", unless otherwise specified, all refer to conventional resuscitation operations in the art, the specific procedures are, for example: when the cells need to be revived, the frozen cells are taken out from the liquid nitrogen and rapidly put into a water bath at 37 ℃ to completely melt the frozen solution within 1-5 min, and the cells are transferred to an ice bath at 4 ℃ after being melted, so that the cell reviving is completed.
The invention discloses a method for separating a large amount of mesenchymal stem cells from an umbilical cord, and the method can be used for preserving the umbilical cord mesenchymal stem cells and establishing an umbilical cord stem cell bank. The inventor of the invention successfully separates a large number of mesenchymal stem cells from umbilical cord by using tissue digestive enzyme to digest umbilical cord tissue blocks and combining an adherence culture method on the basis of summarizing the prior separation culture of mesenchymal stem cells. The mesenchymal stem cells obtained by the method have high purity and a large number, have the same biological characteristics as the bone marrow mesenchymal stem cells, and can be differentiated into osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells and the like. Because the stem cells in the umbilical cord are less immature than the adult stem cells, the content is rich, and the method has wide application prospect in clinic, the mesenchymal stem cells are frozen and stored like umbilical cord blood by using a conventional cell freezing and storing method, and an umbilical cord stem cell bank is established, so that a foundation is laid for the deep research and clinical treatment of the stem cells in the future.
Because the cord blood contains abundant hematopoietic stem cells, people store the important biological resource of the cord hematopoietic stem cells of dogs, and a treatment means is provided for various blood system diseases and immune system diseases. The umbilical cord mesenchymal stem cells are used as a more important stem cell resource, and are frozen in the deep low-temperature liquid nitrogen at the temperature of-196 ℃ for long-term storage by using a conventional cell freezing method, so that an umbilical cord stem cell bank is established, and seeds are stored for stem cells in the future or for application of treatment.
According to the method, the medium formula of the intermediate mesenchymal stem cells can successfully and effectively perform in-vitro expansion on umbilical mesenchymal stem cells. According to the method of the invention, the setting of the fluid change and tissue removal times shortens the time for the adherent cells to reach the specified fusion rate. According to the method of the invention, the formulation of digestive enzymes and the digestion time and method of umbilical cord tissue can successfully and effectively separate whole cells from the tissue.
The invention has simple operation, convenience and practicability, can obtain a large number of mesenchymal stem cells, has good differentiation performance, and has the capability of differentiating into cells such as osteoblasts, adipocytes, chondrocytes, endothelial cells, nerve cells and the like. The invention successfully separates and obtains a large number of mesenchymal stem cells with higher purity from the umbilical cord of the dog, and can establish an umbilical cord stem cell bank of the dog to store the stem cells with great application prospect by using the method. The method is simple and easy to implement, and the umbilical cord is the same as cord blood, so that the cell components are relatively naive, the source is wide, the method is convenient and easy to obtain, and the method has wide prospect in the application of stem cell canine animals.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
EXAMPLE 1 preparation of mesenchymal Stem cells from the umbilical cord of canine
(1) Sterilizing and cleaning: in a biosafety cabinet, sterilizing the surface of the umbilical cord tissue (fresh in-vitro umbilical cord tissue) of the dog by using a sterilizing solution (75% ethanol), cutting off the umbilical cord, spreading (spreading on a culture plate with the diameter of 10 cm), and cleaning the umbilical cord tissue by using a buffer solution (0.025M sodium dihydrogen phosphate buffer solution with the pH of 6.5) so as to reduce red blood cells on the umbilical cord tissue;
(2) Digestion treatment: cutting umbilical cord tissue obtained in the step (1) into tissue blocks (cubic blocks with the volume ratio of 1:1) in another cell culture plate, putting the tissue blocks into a digestive enzyme solution (preparation method comprises the steps of adding 0.1g of type I collagenase into 100ml of DMEM-F12, filtering with a 0.45 mu m filter to obtain digestive juice), performing digestion for 0.5-3 hours (in the example, digestion for 1.5 hours at 37 ℃), filtering to remove the tissue blocks (in the example, filtering with a 100 mu m filter screen), adding a mesenchymal stem cell culture medium (added in the volume ratio of 1:1; in the example, the mesenchymal stem cell culture medium contains 15 parts by weight of FBS, 1 part by weight of L-Glutamine, 0.05 parts by weight of Gentamicin and 84 parts by weight of DMEM-F12) to terminate digestion, performing cell cleaning on cells obtained by digestion (in the example, removing supernatant at 1250rpm for 1250 minutes, adding a centrifugal buffer, re-suspending cells at 1250rpm, removing supernatant for 10 minutes, and obtaining a final cell suspension sample of cell suspension, and performing cell suspension counting;
(3) Cell culture: placing the cell suspension obtained in step (2) into a culture vessel (in this example, at a density of about 1X 10 4 /cm 2 Added), and then the culture vessel is put into an incubator (CO 2 The concentration is 5%, the temperature is 37 ℃) and culturing is carried out, when the culturing is carried out until the 2 nd to 7 th days (in this example, the culturing is carried out generally until the 4 th day), the culture container is taken out from the incubator, a proper amount (3 ml) of mesenchymal stem cell culture medium is added, and the culturing is continued; will be cultured on days 8-11 (in this example, day 9)Taking the container out of the incubator, performing the first full liquid exchange, and continuing culturing; the liquid is completely changed every 1-3 days (2 days in this example) after the process;
(4) Cell passage: after the adherent cell fusion rate in the culture vessel reached 40% -70% (60% in this example), the cells were fused with digestive enzymes (TrypLE in this example) TM Express) separating the adherent cells from the bottom of the container, centrifuging, pumping out the supernatant, adding a mesenchymal stem cell culture medium to re-suspend the cells, inoculating the cells into the culture container for culture, and changing the liquid once every 1-3 days (in this example, every 2 days) until the fusion rate reaches 70-90% (in this example, 80%), thus obtaining the P1-generation umbilical cord mesenchymal stem cells; then the necessary passage (in this case, passage to P15 generation, wherein the purity of the cells after 3 generation passage is more than 95%; in this case, commercial product TrypLE TM Express has the following composition: 200.0mg/L of potassium chloride, 200.0mg/L of potassium dihydrogen phosphate, 8000.0mg/L of sodium chloride, 2160.0mg/L of disodium hydrogen phosphate heptahydrate, EDTA457.6mg/L and commercial amounts of rProtease; the TrypLE TM Express is commercially available from the Simer Feier company, see http:// www.thermofisher.com/cn/zh/home/technical-resources/media-formulation.346.Html for information regarding technology;
(5) Freezing: and (3) adding a cell freezing solution (volume ratio is 1:1) into the umbilical cord mesenchymal stem cells obtained in the step (4), and freezing in liquid nitrogen for later use (in this example, the cell freezing solution comprises 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide and 15 parts of canine blood albumin). Before freezing, detailed information (including canine age, canine seeds, genetic information, vaccination information, virus detection information and the like) of the mesenchymal stem cells is input into a computer database, and a data archive is established for future reference.
EXAMPLE 2 preparation of mesenchymal Stem cells from umbilical cord of canine
(1) Sterilizing and cleaning: in a biosafety cabinet, sterilizing the surface of the umbilical cord tissue (fresh in-vitro umbilical cord tissue) of the dog by using a sterilizing solution (75% ethanol), cutting off the umbilical cord, spreading (spreading on a culture plate with the diameter of 10 cm), and cleaning the umbilical cord tissue by using a buffer solution (0.025M sodium dihydrogen phosphate buffer solution with the pH of 6.5) so as to reduce red blood cells on the umbilical cord tissue;
(2) Digestion treatment: cutting umbilical cord tissue obtained in the step (1) into tissue blocks (cubic blocks with the volume ratio of 1:1) in another cell culture plate, putting the tissue blocks into a digestive enzyme solution (preparation method comprises the steps of adding 0.1g of type I collagenase into 100ml of DMEM-F12, filtering with a 0.45 mu m filter to obtain digestive juice), digesting for 0.5-3 hours (in the example, digesting for 1.5 hours at 37 ℃), filtering to remove the tissue blocks (in the example, filtering with a 100 mu m filter screen), adding a mesenchymal stem cell culture medium (added in the volume ratio of 1:1), in the example, containing 15 parts by weight of FBS, 1 part by weight of L-Glutamine and 0.05 parts by weight of Gentamicin, 84 parts by weight of DMEM-F12 and 0.025% w/v glycine) to terminate digestion, performing cell washing on cells obtained by digestion (in the example, centrifuging at 1250rpm for 10 minutes, adding 1250rpm to remove supernatant, centrifuging at 1250rpm, and obtaining a small cell suspension sample of cell suspension after the cell suspension is removed (in the example, cell suspension is extracted);
(3) Cell culture: placing the cell suspension obtained in step (2) into a culture vessel (in this example, at a density of about 1X 10 4 /cm 2 Added), and then the culture vessel is put into an incubator (CO 2 The concentration is 5%, the temperature is 37 ℃) and culturing is carried out, when the culturing is carried out until the 2 nd to 7 th days (in this example, the culturing is carried out generally until the 4 th day), the culture container is taken out from the incubator, a proper amount (3 ml) of mesenchymal stem cell culture medium is added, and the culturing is continued; taking the culture container out of the incubator at 8-11 days (in this example, 9 days), performing the first full liquid exchange, and continuing culturing; the liquid is completely changed every 1-3 days (2 days in this example) after the process;
(4) Cell passage: after the adherent cell fusion rate in the culture vessel reached 40% -70% (60% in this example), the cells were fused with digestive enzymes (TrypLE in this example) TM Express, 1, 2-propanediol with additional addition of 0.05% w/v is added into the digestive enzyme to separate the adherent cells from the bottom of the container, centrifugate, extract supernatant, add the culture medium of mesenchymal stem cells to re-suspend the cells, and inoculate the culture container for culture, and then change every 1-3 days (in this example, every 2 days)The liquid is used once until the fusion rate reaches 70-90% (80% in this example), and the P1 generation umbilical cord mesenchymal stem cells are obtained; then the necessary passaging was carried out according to the above-mentioned culture method (in this case, passaging to the P15 generation, wherein the purity of the cells after passaging by 3 generation was more than 95%; in this case, trypLE was used) TM Express has the following composition: 200.0mg/L of potassium chloride, 200.0mg/L of potassium dihydrogen phosphate, 8000.0mg/L of sodium chloride, 2160.0mg/L, EDTA 457.6mg/L of disodium hydrogen phosphate heptahydrate and commercial amounts of rProtease; the TrypLE TM Express is commercially available from the Simer Feier company, see http:// www.thermofisher.com/cn/zh/home/technical-resources/media-formulation.346.Html for information regarding technology;
(5) Freezing: and (3) adding a cell freezing solution (volume ratio is 1:1) into the umbilical cord mesenchymal stem cells obtained in the step (4), and freezing in liquid nitrogen for later use (in this example, the cell freezing solution comprises 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide and 15 parts of canine blood albumin). Before freezing, detailed information (including canine age, canine seeds, genetic information, vaccination information, virus detection information and the like) of the mesenchymal stem cells is input into a computer database, and a data archive is established for future reference.
In example 1, the average yield per gram of umbilical cord tissue of the P1-generation canine mesenchymal stem cells obtained from step (1) to step (4) was 3 to 7X 10 by 5 experiments 5 In the cell range (when the human umbilical cord tissue is treated in the same manner as in example 1 of the present invention, the average yield per gram of umbilical cord tissue can be 3 to 6X 10) 7 Cell range, two orders of magnitude larger, possibly due to species differences). In this example 2, the average yield per gram of umbilical cord tissue of the P1-generation canine mesenchymal stem cells obtained in the steps (1) to (4) was 4 to 9X 10 7 Cell range (average result from 5 experiments, the same applies below). In the supplemental test of reference example 2, when glycine was not added to the mesenchymal stem cell medium, the average yield per gram of umbilical cord tissue of the P1-generation canine umbilical cord mesenchymal stem cells was 2 to 4X 10 5 Cell range. In the supplemental test of reference example 2, the enzyme TrypLE was digested TM Without supplementing 1, 2-propanediol in Express, the umbilical cord mesenchymal stem of the P1-generation dog is fineThe average yield of cells per gram of umbilical cord tissue is 3-5×10 5 A cell range; this suggests that glycine was only supplemented in the mesenchymal stem cell medium, while in the digestive enzyme TrypLE TM When 1, 2-propylene glycol is added in Express, the yield of the canine umbilical cord mesenchymal stem cells can be remarkably improved.
In the present invention, trypan blue staining, which is conventional in the art, is used for determining the number of cells and/or the viability of cells, unless otherwise indicated.
The canine umbilical cord mesenchymal stem cells obtained in the above examples 1 to 2 of the present invention were frozen with a conventional frozen stock solution (formulation: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin) for 30 days, then resuscitated with a conventional method, and the number of cells before freezing and the number of cells after resuscitated were measured, and the percentage obtained by dividing the number of cells after resuscitating by the number of cells before freezing and multiplying 100% was used as the survival percentage. The percent survival of the cryopreserved-resuscitated process for the P1 to P15 generations obtained in example 1 is in the range of 67-75%, e.g., the percent survival of the P3 generation canine umbilical cord mesenchymal stem cells of example 1 is 72%; the percent survival of the cryopreserved-resuscitated process for the P1 to P15 passages obtained in example 2 is in the range of 65 to 76%, e.g., the percent survival of the P5 passage canine umbilical cord mesenchymal stem cells of example 2 is 68%.
Cryopreservation and resuscitation of mesenchymal stem cells conventional in the art is performed according to a method comprising the steps of: uniformly mixing the prepared mesenchymal stem cells with cell freezing solution, and then freezing the cell solution in liquid nitrogen until the cells are used; when the cells need to be revived, the frozen cells are taken out from the liquid nitrogen and rapidly put into a water bath at 37 ℃ to completely melt the frozen solution within 1-5 min, and the cells are transferred to an ice bath at 4 ℃ after being melted, so that the cell reviving is completed. In the invention, unless otherwise indicated, both freezing and thawing are carried out according to the methods described above.
Example 3: referring to example 1, but in step (5), modified cell cryopreservation solution (comprising 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin and 0.25% w/v dextran-40) was used instead, and the results showed that the survival percentage of the P1 to P15 generation cells through the cryopreservation-resuscitation process was in the range of 92-96%, for example, the survival percentage of the P3 generation canine umbilical cord mesenchymal stem cells was 93%.
Example 4: referring to example 2, but in step (5) modified cell cryopreservation solution (comprising 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin, 0.25% w/v dextran-40) was used, the results showed that the survival percentage of P1 to P15 generation cells through the cryopreservation-resuscitation process was in the range of 91-97%, for example, the survival percentage of P5 generation canine umbilical cord mesenchymal stem cells was 94%. Surprisingly, it has been found that the addition of a minor amount of dextran to the cryopreservation solution significantly increases the cell death caused during the cell cryopreservation-resuscitation process.
In the present invention, the preparation methods of the various cell cryopreservation solutions are prepared according to the following methods, unless otherwise specified: uniformly mixing the DMEM-F12, dimethyl sulfoxide and canine blood albumin in a specified proportion by volume, and optionally adding other specified components in specified amounts to dissolve the components.
Example 5: referring to example 1, but in step (5), another modified cell cryopreservation solution (which may be referred to as cell cryopreservation solution B1, comprising 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin, 0.07% w/v povidone K30 and 0.6% w/v histidine) was used instead, and after freezing in liquid nitrogen for 30 days, it was revealed that the survival percentage of P1 to P15 generation cells through the cryopreservation-resuscitating process was in the range of 93 to 95%, for example, the survival percentage of P3 generation canine umbilical cord mesenchymal stem cells was 93%.
Example 6: referring to example 2, but in step (5), another modified cell cryopreservation solution (which may be referred to as cell cryopreservation solution B1, comprising 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin, 0.07% w/v povidone K30 and 0.6% w/v histidine) was used instead, and after freezing in liquid nitrogen for 30 days, it was revealed that the survival percentage of P1 to P15 generation cells through the cryopreservation-resuscitating process was in the range of 92 to 95%, for example, the survival percentage of P5 generation canine umbilical cord mesenchymal stem cells was 93%.
Example 7: referring to example 1, but in step (5), another modified cell cryopreservation solution (which may be referred to as cell cryopreservation solution B2, comprising 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin, 0.06% w/v povidone K30 and 0.7% w/v histidine) was used instead, and after 30 days of cryopreservation in liquid nitrogen, it was revealed that the survival percentage of P1 to P15 generation cells through the cryopreservation-resuscitating process was 93 to 96%, for example, the survival percentage of P3 generation canine umbilical cord mesenchymal stem cells was 94%.
Example 8: referring to example 1, but in step (5), another modified cell cryopreservation solution (which may be referred to as cell cryopreservation solution B3, comprising 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine blood albumin, 0.08% w/v povidone K30 and 0.5% w/v histidine) was used instead, and after 30 days of cryopreservation in liquid nitrogen, it was revealed that the survival percentage of P1 to P15 generation cells through the cryopreservation-resuscitating process was 93 to 96%, for example, the survival percentage of P3 generation canine umbilical cord mesenchymal stem cells was 95%.
Experimental example 1 stability after thawing of cryopreserved cells
The cells recovered after 30 days of freezing in liquid nitrogen by using the corresponding freezing solution in examples 1 to 8 are placed at the temperature of 25+/-1 ℃ for 8 hours without changing the solution, the viable cell numbers in the unit volume of the cell solution before and after the treatment of the cells for 25-8 hours are respectively measured by trypan blue staining, and the quotient of the viable cell number at 8 hours divided by the viable cell number at 0 hour is multiplied by 100 percent to obtain the residual percentage of the cells after the treatment for 25-8 hours. As a result, the percentages of cell residues in examples 1 to 4 were 42.3%, 45.6%, 38.7% and 44.1%, respectively; the residual percentages of cells in examples 5 to 8 were 91.4%, 88.7%, 90.2% and 89.3%, respectively.
Experimental example 2 stability after thawing of cryopreserved cells
The cells recovered after 30 days of freezing in liquid nitrogen by using the corresponding freezing solution in examples 1 to 8 are placed at the temperature of 4+/-1 ℃ for 96 hours without changing the solution, the viable cell numbers in the unit volume of the cell solution before and after the treatment of the cells for 4-96 hours are respectively measured by trypan blue staining, and the quotient of the viable cell number at 96 hours divided by the viable cell number at 0 hour is multiplied by 100 percent to obtain the residual percentage of the cells after the treatment for 4-96 hours. As a result, the percentages of cell residues in examples 1 to 4 were 47.4%, 44.5%, 52.3% and 42.7%, respectively; the residual percentages of the cells in examples 5 to 8 were 90.2%, 88.6%, 91.3% and 89.4%, respectively.
According to the results of the experimental examples 1-2, when the cell cryopreservation solutions B1-B3 are used, the recovered cells can survive longer than the cells in other cryopreservation solutions obviously under the condition of not changing the solution, and the application occasion and the application environment with remarkably stronger adaptability are provided for the mesenchymal stem cells.
Supplementary example 9: referring to examples 5 to 8, respectively, but without addition of povidone to the used cell cryopreservation solution, cells were resuscitated after 30 days of cryopreservation, and the residual percentage was determined/calculated by referring to the method of experimental example 1; as a result, the percentages of cell residues in examples 5 to 8, but without addition of povidone, were 44.3%, 41.8%, 46.2% and 39.7%, respectively. Supplementary example 10: referring to examples 5 to 8, respectively, but histidine was not added to the cell frozen stock solution used, and the cells were resuscitated after 30 days of freezing, and the residual percentage was measured/calculated by referring to the method of experimental example 1; as a result, the percentages of cell residues in examples 5 to 8, but without histidine addition, were 42.2%, 40.6%, 39.6% and 37.8%, respectively. Supplementary example 11: referring to examples 5 to 8, respectively, but without addition of povidone to the used cell cryopreservation solution, cells were resuscitated after 30 days of cryopreservation, and the residual percentage was determined/calculated by referring to the method of experimental example 2; as a result, the cell residue percentages in examples 5 to 8, but without addition of povidone, were 38.7%, 44.3%, 42.7% and 41.5%, respectively. Supplementary example 12: referring to examples 5 to 8, respectively, but histidine was not added to the cell frozen stock solution used, and the cells were resuscitated after 30 days of freezing, and the residual percentage was measured/calculated by referring to the method of experimental example 2; as a result, the percentages of cell residues in examples 5 to 8, but without histidine addition, were 45.7%, 44.5%, 42.3% and 45.3%, respectively. From these results, it can be seen that cells after resuscitation exhibit excellent stability only when povidone and histidine are included in the cell cryopreservation solution.
Experimental example 3 biological characterization of umbilical cord MSC
The following experiments were performed on the canine umbilical cord mesenchymal stem cells obtained in example 1 and example 2, and the results of the experiments on the two stem cells were not different.
1. Cell growth and morphological characteristics thereof
Through the isolated culture of example 1 and example 2, the obtained dog umbilical cord mesenchymal stem cells can obviously see spindle-shaped adherent cells under a microscope after 48 hours of culture, and can form vortex-shaped cell clones about 8 days, and can form an adherent layer with about 70% fusion after digestion and passage. In the culture process, the cell has relatively uniform morphology, fast proliferation speed, fast adherence speed, easy digestion by pancreatin, passage to 3-15 generations and no obvious change in morphology and growth characteristics.
2. Flow cytometry identification of MSC surface markers
And taking the 1 st, 3 rd, 5 th, 10 th and 15 th generation cells respectively, detecting cell surface markers by flow cytometry, and dynamically observing the change of the cell surface markers in the culture process. The cell phenotype detection result shows that the markers CD44, CD90 and CD105 special for the cell surface mesenchymal stem cells do not express CD11, CD19 and CD34.
3. Drawing umbilical cord MSC growth curve and measuring multiplication time in logarithmic growth phase
Taking logarithmic growth phase cells, performing digestion and counting, and preparing cell suspension (2×10) from LG-DMEM medium containing 10% FBS 4 Per ml), 0.5ml,37℃and 5% CO per well in a 24-well plate 2 Culturing under saturated humidity. 3 wells per day were taken, the number of viable cells counted after trypan blue staining, the average calculated, and observation was continued for 7 days. And drawing a cell growth curve by taking the culture time as the horizontal axis and the cell number as the vertical axis. The doubling time of the cells in the logarithmic growth phase, td=tlig2/Lg (Nt/No), td, was calculated by the Patterson formula: doubling time (h), T: time (h) taken for the cells to increase from No to Nt, N: cell number.
The doubling time was calculated by plotting the cell growth curve from the results of daily cell counts. As can be seen from the cell growth curve, the cells were in exponential growth phase on days 3-5. And calculating the doubling time of the 5 th generation cells in the exponential growth phase within the range of 22-36 hours according to the formula.
4. Identification of Multi-way differentiation potential of umbilical cord MSC
(1) Osteogenesis induction: 3 generations of MSC, according to 5×10 4 Inoculating six-hole plate at 37deg.C with 5% CO 2 Culturing in MSC culture medium under saturated humidity for 24 hr, replacing DMEM-HG containing 10% of selected FBS, adding dexamethasone 0.1 μm, ascorbyl phosphate 50 μm, beta-phosphoglyceride 5mM, and placing at 37deg.C and 5% CO 2 Culturing under saturated humidity, and changing liquid every 3 days for 2-4 weeks. Alkaline phosphatase staining identified osteoblast formation and Von Kossa staining identified bone nodule formation. After culturing in DMEM-HG containing 10% of the selected FBS at 0.1. Mu.M with dexamethasone, 50. Mu.M with ascorbyl phosphate and 5mM with beta-phosphoglycerate for 1 week, the cell morphology was changed markedly from spindle-shaped fibroblast-like to polygonal, similar to neuron-like cell-like, and filament-like protrusions were present around the cell periphery and were extendable to the periphery. After further culturing for more than 2 weeks, calcified spots appear in the cell matrix, mineralization gradually appears, and a multi-layer nodule structure starts to form, and obvious calcified nodules are visible after culturing for 4 weeks. The phosphatase staining was strongly positive for 2 weeks Shi Jianxing, reaching 92% or more, whereas the uninduced control group was mostly negative, with less than 5% showing weak positivity, indicating that the cells had transformed into osteoblasts. von Kossa staining can stain the calcium deposited in the bone nodules black, the induced group sees a large number of black bone nodules with a clear steric structure, while the control group has no positive response at any time.
(2) Fat induction: 3 generations of MSC, according to 5×10 4 Inoculating into six-hole plate, placing at 37deg.C and 5% CO 2 Culturing in MSC culture medium for 24 hr under saturated humidity, replacing high sugar DMEM containing 10% of selected FBS, adding dexamethasone 0.5 μm, indomethacin 25 μ M, IBMX 0.5.5 mM, insulin 2 μg/ml, standing at 37deg.C, and 5% CO 2 Culturing under saturated humidity, changing liquid every 3 days for half a period of time, and co-inducing for 2 weeks, and performing oil red staining to identify lipid drop formation. Adding dexamethasone 0.5 μm into DMEM-HG containing 10% of screened FBS, and relieving inflammation50 mu M, IBMX 0.5.5 mM, 5 mu g/ml insulin for 3 days, the cells are changed in morphology, the cells gradually shrink and shorten from spindle-shaped fibroblast-like, and more than 90% of the cells become cubic or polygonal; the continuous culture is carried out for 7 days, tiny lipid droplets appear in the cells under the microscope, the lipid droplets gradually increase and fuse along with the extension of the culture time, and when the culture is carried out for 2 weeks, the whole cells are filled with the fused lipid droplets. Oil red O staining can be seen as specific staining of intracellular produced fat to red.
The above-described test was performed on the canine umbilical cord mesenchymal stem cells recovered after 30 days of freezing in examples 3 to 8, and the results were not different from the test results of the two stem cells of examples 1 and 2.
The detection of a series of data indexes shows that the canine mesenchymal stem cells separated by the method have the capability of differentiating into osteoblasts, adipocytes and the like, and the canine mesenchymal stem cells obtained by the method have stem cell characteristics.
The present inventors have made experiments with reference to the examples and experimental examples in the section "detailed description" described above, except that canine umbilical cord was replaced with canine fat, canine adipose mesenchymal stem cells could be obtained efficiently as in the canine umbilical cord results described above, and the obtained canine adipose mesenchymal stem cells exhibited the same properties as in the experimental examples described above.
In the supplementary test, the present inventors refer to the examples and experimental examples of the "detailed description" section herein above, except that the canine umbilical cord was replaced with the feline umbilical cord, and the feline umbilical cord mesenchymal stem cells could be obtained similarly efficiently as in the canine umbilical cord results described above, and the obtained feline umbilical cord mesenchymal stem cells exhibited the same properties as in the experimental examples described above.
The present inventors have made experiments with reference to the examples and experimental examples in the section "detailed description" described above, except that the canine umbilical cord was replaced with the feline fat, and the feline adipose-derived mesenchymal stem cells could be obtained similarly efficiently as in the canine umbilical cord results described above, and the obtained feline adipose-derived mesenchymal stem cells exhibited the same properties as in the experimental examples described above.
The method is simple and stable to operate, and the prepared fat or umbilical cord mesenchymal stem cells have high activity and can be used for treating refractory diseases of dogs or cats by conventional means.

Claims (8)

1. A method of cryopreserving and resuscitating canine umbilical cord-derived mesenchymal stem cells, the method comprising the steps of:
(51) The prepared mesenchymal stem cells derived from the dog umbilical cord and the cell freezing solution are mixed according to the volume ratio of 1:1, then placing the cell fluid in liquid nitrogen for freezing, and preserving until the cells are used; the composition of the cell freezing solution is as follows: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.06-0.08% w/v povidone K30 and 0.5-0.7% w/v histidine;
(52) When the cells need to be revived, taking out the frozen cells from the liquid nitrogen, rapidly putting the frozen cells into a water bath at 37 ℃, completely thawing the frozen solution within 1-5 min, and transferring the thawed cells to an ice bath at 4 ℃ to finish cell reviving;
wherein, the mesenchymal stem cells of dog umbilical cord source are prepared by the following method:
(1) Sterilizing and cleaning: sterilizing the surface of the umbilical cord tissue of the dog by using a sterilizing liquid, shearing and spreading the umbilical cord, and cleaning the umbilical cord tissue by using a buffer solution to reduce red blood cells on the umbilical cord tissue;
(2) Digestion treatment: cutting umbilical cord tissue obtained in the step (1) into tissue blocks, putting the tissue blocks into a digestive enzyme solution, performing digestion treatment for 0.5-3 hours, filtering to remove the tissue blocks, adding a mesenchymal stem cell culture medium to terminate digestion, and then performing cell cleaning on cells obtained by digestion to finally obtain a cell suspension;
(3) Cell culture: placing the cell suspension obtained in the step (2) into a culture container, placing the culture container into an incubator for culture, taking the culture container out of the incubator when the culture is carried out for 2-7 days, supplementing a proper amount of mesenchymal stem cell culture medium, and continuing to culture; taking the culture container out of the incubator at 8-11 days, performing the first full liquid exchange, and continuing culturing; performing full liquid change every 1-3 days;
(4) Cell passage: separating the adherent cells from the bottom of the culture container by using digestive enzyme after the fusion rate of the adherent cells in the culture container reaches 40% -70%, centrifuging, pumping out supernatant, adding a mesenchymal stem cell culture medium to re-suspend the cells, inoculating the cells into the culture container for culture, and changing the liquid once every 1-3 days until the fusion rate reaches 70% -90%, thereby obtaining the P1-generation umbilical cord mesenchymal stem cells; the necessary passages were then carried out according to the above-described culture method.
2. The method of claim 1, wherein the composition of the cell cryopreservation solution is: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.07% w/v povidone K30 and 0.6% w/v histidine.
3. A cell cryopreservation solution for cryopreservation and resuscitation of canine umbilical cord-derived mesenchymal stem cells, wherein the composition is as follows: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.06-0.08% w/v povidone K30 and 0.5-0.7% w/v histidine.
4. A cell cryopreservation solution according to claim 3 wherein the composition is: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.07% w/v povidone K30 and 0.6% w/v histidine.
5. The cell cryopreservation solution according to claim 4, wherein the cryopreservation and resuscitation of mesenchymal stem cells comprises the steps of:
(51) The prepared mesenchymal stem cells derived from the dog umbilical cord and the cell freezing solution are mixed according to the volume ratio of 1:1, then placing the cell fluid in liquid nitrogen for freezing, and preserving until the cells are used;
(52) When the cells need to be revived, taking out the frozen cells from the liquid nitrogen, rapidly putting the frozen cells into a water bath at 37 ℃, completely thawing the frozen solution within 1-5 min, and transferring the thawed cells to an ice bath at 4 ℃ to finish cell reviving;
Wherein, the mesenchymal stem cells of dog umbilical cord source are prepared by the following method:
(1) Sterilizing and cleaning: sterilizing the surface of the umbilical cord tissue of the dog by using a sterilizing liquid, shearing and spreading the umbilical cord, and cleaning the umbilical cord tissue by using a buffer solution to reduce red blood cells on the umbilical cord tissue;
(2) Digestion treatment: cutting umbilical cord tissue obtained in the step (1) into tissue blocks, putting the tissue blocks into a digestive enzyme solution, performing digestion treatment for 0.5-3 hours, filtering to remove the tissue blocks, adding a mesenchymal stem cell culture medium to terminate digestion, and then performing cell cleaning on cells obtained by digestion to finally obtain a cell suspension;
(3) Cell culture: placing the cell suspension obtained in the step (2) into a culture container, placing the culture container into an incubator for culture, taking the culture container out of the incubator when the culture is carried out for 2-7 days, supplementing a proper amount of mesenchymal stem cell culture medium, and continuing to culture; taking the culture container out of the incubator at 8-11 days, performing the first full liquid exchange, and continuing culturing; performing full liquid change every 1-3 days;
(4) Cell passage: separating the adherent cells from the bottom of the culture container by using digestive enzyme after the fusion rate of the adherent cells in the culture container reaches 40% -70%, centrifuging, pumping out supernatant, adding a mesenchymal stem cell culture medium to re-suspend the cells, inoculating the cells into the culture container for culture, and changing the liquid once every 1-3 days until the fusion rate reaches 70% -90%, thereby obtaining the P1-generation umbilical cord mesenchymal stem cells; the necessary passages were then carried out according to the above-described culture method.
6. A method of preparing mesenchymal stem cells from the umbilical cord of a canine and cryopreserving, the method comprising the steps of:
(1) Sterilizing and cleaning: sterilizing the surface of the umbilical cord tissue of the dog by using a sterilizing liquid, shearing and spreading the umbilical cord, and cleaning the umbilical cord tissue by using a buffer solution to reduce red blood cells on the umbilical cord tissue;
(2) Digestion treatment: cutting umbilical cord tissue obtained in the step (1) into tissue blocks, putting the tissue blocks into a digestive enzyme solution, performing digestion treatment for 0.5-3 hours, filtering to remove the tissue blocks, adding a mesenchymal stem cell culture medium to terminate digestion, and then performing cell cleaning on cells obtained by digestion to finally obtain a cell suspension;
(3) Cell culture: placing the cell suspension obtained in the step (2) into a culture container, placing the culture container into an incubator for culture, taking the culture container out of the incubator when the culture is carried out for 2-7 days, supplementing a proper amount of mesenchymal stem cell culture medium, and continuing to culture; taking the culture container out of the incubator at 8-11 days, performing the first full liquid exchange, and continuing culturing; performing full liquid change every 1-3 days;
(4) Cell passage: separating the adherent cells from the bottom of the culture container by using digestive enzyme after the fusion rate of the adherent cells in the culture container reaches 40% -70%, centrifuging, pumping out supernatant, adding a mesenchymal stem cell culture medium to re-suspend the cells, inoculating the cells into the culture container for culture, and changing the liquid once every 1-3 days until the fusion rate reaches 70% -90%, thereby obtaining the P1-generation umbilical cord mesenchymal stem cells; then carrying out necessary passage according to the culture method;
(5) Freezing: in the volume ratio of 1:1, adding a cell freezing solution into the umbilical cord mesenchymal stem cells obtained in the step (4), and freezing in liquid nitrogen for later use; the composition of the cell freezing solution is as follows: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.06-0.08% w/v povidone K30 and 0.5-0.7% w/v histidine.
7. The method according to claim 6, wherein the composition of the cell cryopreservation solution is: 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide, 15 parts of canine albumin, 0.07% w/v povidone K30 and 0.6% w/v histidine.
8. The method according to claim 6, wherein CO in the incubator of step (3) 2 The concentration was 5%, and the incubator temperature was controlled at 37 ℃.
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