CN116941606B - Construction method of cell tissue library of umbilical cord tissue and related products and application thereof - Google Patents

Construction method of cell tissue library of umbilical cord tissue and related products and application thereof Download PDF

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CN116941606B
CN116941606B CN202311210336.6A CN202311210336A CN116941606B CN 116941606 B CN116941606 B CN 116941606B CN 202311210336 A CN202311210336 A CN 202311210336A CN 116941606 B CN116941606 B CN 116941606B
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umbilical cord
cord tissue
cell
tissue
cells
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CN116941606A (en
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武晓云
王黎明
任宇强
杨宇枭
王煜骏
包宏磊
张宇轩
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Inner Mongolia Yuanyuan 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
    • 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
    • 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
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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

Abstract

The invention discloses a construction method of a cell tissue library of umbilical cord tissue, and related products and applications thereof, wherein the cell tissue library comprises a balancing liquid and a freezing storage liquid, and the balancing liquid comprises the following components: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection; the frozen liquid also comprises DMSO with mass fraction of 5% -10%. The freezing solution and the balancing solution provided by the invention can effectively reduce the damage of direct freezing to umbilical cord tissue blocks and cells thereof, improve the yield and the efficiency of cell resuscitation, and have short culture time period and low library construction cost. And all cell types in the umbilical cord and various bioactive substances in umbilical cord tissues are preserved, so that valuable resources in the umbilical cord are preserved to the greatest extent, and resource waste of samples is avoided.

Description

Construction method of cell tissue library of umbilical cord tissue and related products and application thereof
Technical Field
The invention relates to the technical field of cell tissue library construction, in particular to a construction method of a cell tissue library of umbilical cord tissue, and related products and applications thereof.
Background
Mesenchymal Stem Cells (MSCs), which are also called mesenchymal stromal cells, belong to one type of adult stem cells. Because MSCs are widely available and easy to separate, can be rapidly amplified, and have unique immunoregulatory properties, the MSCs become ideal choices in the field of transplantation and for the treatment of immune diseases. MSCs have provided promise for the treatment of a variety of inflammation-related disorders from the results of an increasing number of animal experiments and clinical trials. From the aspects of stem cell record projects and new medicine declaration projects in China, MSCs are the most studied and applied stem cell types in the current stem cell treatment field, and more than 60% of MSCs (UCMSCs) from perinatal umbilical cord tissue sources are adopted. The perinatal umbilical cord tissue is medical waste, is discarded more than needed, and has the advantages of abundant sources, convenient collection, no ethical disputes, less influence of diseases and the like. At present, almost all UCMSCs have been found to have high proportion, low immunogenicity, strong proliferation and differentiation capability, stronger immunoregulation capability and easy mass preparation, and have been increasingly favored by scientists and clinicians. Couto et al reported that UCMSCs were the dominant perinatal cell type in clinical trials, and umbilical cord tissue was the major source of MSCs in all MSCs clinical trials since 2016 (Couto PS, shapirishili G, bersenev A, verter F, first decade of clinical trials and published studies with mesenchymal stromal cells from umbilical cord tisue. Regen Med 2019;14 (4): 309-319). UCMSCs are becoming a favored source of stem cells in new drug development and clinical documentation. The stem cell inventory project and the new medicine declaration project of the product on the market of the global stem cells in China all relate to various different indications of UCMSCs. UCMSCs also show good curative effect in the treatment of severe patients with new coronaries in the early 2020. International UCMSCs therapeutic products are also accelerating the pace of market entry.
Modern researches have found that neonatal umbilical cord tissues contain abundant stem cells and various bioactive substances. The umbilical cord tissue contains MSCs as the main source and other stem cells, such as endothelial progenitor cells. This is also an important stem cell biological resource. Besides the clinical value of various active cells, the natural tissue structure in the tissues and important active components such as various nucleic acid and protein molecules such as hormone and the like are accompanied, and the method has important significance in diagnosis, treatment and scientific research of clinical diseases. The neonate accompanying organization is an important carrier of neonatal vital information, is also an initial vital information source with great medical value, and is an important vital characteristic information source for personalized medical treatment and health maintenance, and the foundation for constructing ethnic group health big data is the same. The stored accompanying tissue sampling is used for analyzing different kinds of initial health information, so that important decision basis is provided for prediction, prevention and treatment of various common diseases, and the method is also an important content of future health data.
The stem cell bank is a place and a platform for large-scale collection, preparation, storage and capability of providing stem cells, also called as a 'life bank', and the establishment of the stem cell bank can provide a stable seed source for research application of MSCs. In recent 5 years, various regional stem cell libraries are approved by large and medium cities in each province in China. The domestic stem cell library provides a good platform for the industrialization of MSCs, and also provides high-quality stem cell seeds with legal sources for the clinical application of MSCs, so that the industrialization and the clinical application of MSCs can be promoted strongly.
At present, the method for storing UCMSCs in the industry stem cell library is as follows: separating umbilical cord Wharton's jelly, performing primary and passage amplification culture to a certain amount of UCMSCs, and performing deep low-temperature preservation. Chinese invention patent application number: 202011388141.7 (publication No. CN 112481203A) discloses a construction method of an umbilical cord mesenchymal stem cell seed bank, comprising the following steps: 1) Screening puerpera; 2) Collecting a sample; 3) Detecting a sample; 4) Sample pretreatment; 5) Cell separation and preparation; 6) Primary culture; 7) Subculturing; 8) Cell detection; 9) Freezing and preserving cells; 10 Establishing a cell information file for retrieval; 11 Encode and put in storage and upload the cell information file. This method has several disadvantages: 1) The culture operation is needed, the time period is long, and the preparation cost is high; 2) Other types of cells in the umbilical cord and other nucleic acid, protein molecules such as hormone and other various bioactive substances are discarded as waste, so that the waste of sample resources is caused; 3) The UCMSCs have larger damage after freezing and lower cell efficacy.
Disclosure of Invention
In order to solve the problems, the invention provides a construction method of a cell tissue library of umbilical cord tissue, and related products and applications thereof.
In a first aspect, the present invention provides a cryopreservation protectant comprising a equilibration fluid and a cryopreservation fluid; the balance liquid consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection; the frozen stock solution consists of the following components in the final concentration: 5-10% of DMSO (dimethyl sulfoxide), 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection.
In a second aspect, the present invention provides a cryoprotectant comprising a equilibration fluid and a cryopreservation fluid; the balance liquid consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection; the frozen stock solution consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-10% of DMSO, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection.
In a third aspect, the invention provides a kit for umbilical cord tissue comprising: the cryoprotectant provided in the first or second aspect.
In a fourth aspect, the present invention provides a method for cryopreserving umbilical cord tissue, comprising: freezing and storing the umbilical cord tissue blocks by adopting the freezing and storing protective agent provided in the first aspect or the second aspect: placing the umbilical cord tissue blocks in frozen stock solution for cooling and preserving; or placing the umbilical cord tissue blocks in balance liquid for standing, and then placing in frozen stock solution for cooling and preserving.
In a fifth aspect, the present invention provides a method for constructing a cell tissue library of umbilical cord tissue, comprising: freezing the umbilical cord tissue blocks by adopting the freezing method in the fourth aspect, and resuscitating the frozen umbilical cord tissue blocks.
Specifically, the step of resuscitating the cryopreserved umbilical cord tissue blocks comprises: thawing and dissolving the frozen umbilical cord tissue blocks, and centrifuging to remove supernatant; adding the balancing solution in the first aspect into the centrifuged product, standing for 10-40min, centrifuging to remove supernatant, adding a mixture of the balancing solution and the complete culture medium, standing for 10-40min, centrifuging to remove supernatant, and culturing and amplifying cells in placenta tissue mass to obtain cells.
The invention has the advantages that: the invention optimizes the cryopreservation liquid and the balancing liquid for cryopreserving umbilical cord tissues, and the balancing liquid pretreatment before cryopreservation and/or the balancing liquid pretreatment in the resuscitation process can reduce the damage of freezing to MSCs, improve the yield and the efficiency of cell resuscitation, such as improving the cell doubling capacity, enhancing the lymphocyte proliferation inhibition capacity, improving the immunoregulation function and the like.
The cryopreservation reagent and the method provided by the invention can be used for directly cryopreserving the umbilical cord tissue blocks without culture operation, and have the advantages of short time period and low library construction cost. But also saves all cell types in the umbilical cord (including but not limited to human umbilical cord mesenchymal stem cells, sub-totipotent stem cells, hematopoietic stem/progenitor cells, endothelial progenitor cells, immune cells and the like) and various bioactive substances such as nucleic acid, protein molecules such as hormone and the like in umbilical cord tissues, saves valuable resources in the umbilical cord to the greatest extent, and avoids the resource waste of samples.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the cell morphology characteristics (x 100) of UCMSCs from three groups in example 7.
FIG. 2 shows the results of in vitro adipogenic induction, osteogenic induction and chondrogenic induction differentiation staining of UCMSCs of the three groups of example 7 (. Times.200).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: a cryopreservation protective agent, which comprises a balancing liquid and a cryopreservation liquid; the balance liquid consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection; the frozen stock solution consists of the following components in the final concentration: 5-10% of DMSO (dimethyl sulfoxide), 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection.
Example 2: a cryopreservation protective agent, which comprises a balancing liquid and a cryopreservation liquid; the balance liquid consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection; the frozen stock solution consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-10% of DMSO, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection.
Example 3: a kit for umbilical cord tissue, comprising: the cryoprotectant provided in example 1 or example 2.
Example 4A method for cryopreserving umbilical cord tissue comprising: the umbilical cord tissue blocks were cryopreserved using the cryopreservation protectant provided in example 1 or example 2: placing the umbilical cord tissue blocks in frozen stock solution for cooling and preserving; or placing the umbilical cord tissue blocks in balance liquid for standing, and then placing in frozen stock solution for cooling and preserving.
In some embodiments, the cooling procedure comprises: the first step: reducing the temperature from room temperature to 0-4 ℃ at a rate of-15 to-5 ℃ per minute; and a second step of: reducing the temperature from 0-4 ℃ to-12 to-4 ℃ at a rate of-5 ℃ per minute; and a third step of: reducing the temperature from-12 to-4 ℃ to-55 to-45 ℃ at a rate of-30 to-20 ℃ per minute; fourth step: regulating the temperature from-55 to-45 ℃ to-18 to-10 ℃ at a speed of 5-15 ℃ per minute; fifth step: regulating the temperature from-18 to-10 ℃ to-50 to-40 ℃ at a rate of-5 to 5 ℃ per minute; sixth step: regulating the temperature from-50 to-40 ℃ to-95 to-80 ℃ at the speed of-15 to-5 ℃ per minute. In the cooling and freezing process of the biological sample, certain heat is released in the phase change period from liquid state to solid state, so that the temperature of the biological sample is raised, and the freezing process without controlling the cooling rate can lead to tissue cell death. The program cooling method is to accurately measure the phase change point of biological sample, and program the cooling program with microcomputer to increase liquid nitrogen input amount during sample phase change, overcome the temperature rise of phase change sample and make cell pass the phase change period safely and fast. The freezing and storing program of the conventional umbilical cord mesenchymal stem cells is that the cooling rate is-1 to-2 ℃/min; when the temperature reaches below minus 25 ℃, the temperature can be increased to minus 5 ℃ to minus 10 ℃/min; at-100℃it can be immersed rapidly in liquid nitrogen. The cooling procedure is superior to conventional cooling, and can more effectively avoid or reduce cell death caused by freezing.
Example 5: a method of constructing a cell tissue library of umbilical cord tissue, comprising: the cryopreservation method described in example 4 was used to cryopreserve the umbilical cord tissue pieces and resuscitate the cryopreserved umbilical cord tissue pieces.
In some embodiments, the step of resuscitating the cryopreserved umbilical cord tissue mass comprises:
thawing and dissolving the frozen umbilical cord tissue blocks, and centrifuging to remove supernatant; adding the balancing solution described in the embodiment 1 into the centrifuged product, standing for 10-40min, centrifuging to remove supernatant, adding a mixture of the balancing solution and a complete culture medium, standing for 10-40min, centrifuging to remove supernatant, and culturing and amplifying cells in placenta tissue blocks to obtain cells.
In some embodiments, the thawing and dissolving step may include: and placing the frozen stock in a constant-temperature water bath at 30-40 ℃ for 1-5 min.
In some embodiments, the step of culturing cells in the placental tissue mass and expanding the cells to obtain cells can be obtained based on prior conventional technical knowledge. In some embodiments, the step comprises: transferring the tissue block into a culture flask, and placing at 37deg.C and 5% CO 2 Culturing in a cell culture box, removing tissue blocks after 4-5 days, and replacing fresh complete culture medium every 3 days. When the cells grew to about 80% confluence, they were grown at 2X 10 3 /cm 2 And carrying out subculture on the density, and obtaining a large number of human umbilical cord mesenchymal stem cells for later use after amplification.
In some embodiments, the centrifugation conditions include: 1400-160 rpm, 5-15 min.
Example 6: frozen stock test
1. Specimen source
The human umbilical cord specimen comes from the term pregnant women of Beijing friendship hospital obstetrics, eliminates fetal malformation, congenital genetic disease and infectious disease history of the women, and signs the informed consent. The donor should be subjected to project examination of human-derived specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.), treponema pallidum, glucose-6-phosphate dehydrogenase and glutamic pyruvic transaminase, and specific requirements are shown in table 1. Collecting umbilical cord tissue under the aseptic condition of an operating room, washing the umbilical cord tissue for 2-3 times by using normal saline, putting the umbilical cord tissue into a collecting bottle, and transporting the umbilical cord tissue to a laboratory for tissue treatment within 12 hours.
TABLE 1 donor requirements
2. Preparation of umbilical cord samples:
cutting off two ends of the umbilical cord, stroking out blood in umbilical cord blood vessels, dividing the umbilical cord into small sections of about 10cm, soaking for 3min by using a disinfectant, and then soaking and washing for 2-3 times by using a washing liquid until no blood stain exists in the washing liquid. Tearing off two arteries and one vein vessel in the umbilical cord by using elbow forceps, cleaning Wharton's jelly for 2-3 times, and shearing until the Wharton's jelly is 1-2 mm 3 The tissue blocks are divided into 8 groups, and 10g of each group is frozen by using different frozen stock solutions respectively.
3. Preparing a frozen stock solution and freezing umbilical cord tissues:
the group 1-7 is prepared by laboratory, and the concentration of DMSO, glycerol, human serum albumin injection and dextran 40 sodium chloride injection in the prepared frozen stock according to the group 1-seventh in the table 2 are all mass fractions. Wharton's jelly tissue is taken and placed in a stock solution containing 2ml of laboratory formulation, and about 2-2.5g of tissue can be placed in each 5ml of stock tube.
Table 2 laboratory concentration of the prepared frozen stock solution
The eighth group is clinical cell therapy grade cryopreservation solution (CryoSure-DEX 40, company OriGen BioMedical, USA, cat# WAK-DEX 40-50-5) meeting the United states pharmacopoeia USP. Wharton's jelly tissue is placed in a clinical cell therapy grade cryopreservation solution (CryoSure-DEX 40, U.S. OriGen BioMedical, inc.: WAK-DEX 40-50-5) containing 2ml of tissue conforming to United states pharmacopoeia USP, and about 2-2.5g of tissue per 5ml of cryopreservation tube may be placed.
CryoSure-DEX40 is a clinical cell therapy grade cryopreservation solution produced by foreign company OriGen BioMedical that meets United states Pharmacopeia USP. The product has the purity of 100%, is produced under the GMP condition, has no pyrogen, endotoxin and mycoplasma, and has higher occupancy rate in the market. Has been applied in the fields of clinical injection, bone marrow transplantation, hematopoietic stem cell separation, cord blood cryopreservation, cornea and other organ preservation, etc.
4. Program cooling and freezing:
after cooling using a programmed cooling instrument (cooling program: step 1: -10: -4.0: -1.0: -8.0: -25.0: -50: -10.0: -14.0: -5: -1.0: -45: -10.0: -90: -10.0: -7: end.), the mixture was placed in liquid nitrogen for 3 months.
5. Tissue resuscitation and cell culture:
taking out the frozen storage tube, placing in a constant-temperature water bath box at 37 ℃ for 3 minutes, centrifuging at 1500r/min for 5 minutes, removing the frozen storage liquid, rapidly transferring the tissue suspension into a sterile centrifuge tube containing a culture medium (IMDM basal medium+5% platelet lysate), centrifuging and washing for 2-3 times, transferring the tissue block into a T25 culture flask, placing in a temperature of 37 ℃ and 5% CO 2 Culturing in a cell incubator, removing tissue blocks after 5 days, changing fresh medium every 3 days, and culturing at 2×10 when cells grow to about 80% confluence 3 /cm 2 Subculturing is carried out at the density.
6. Number detection of umbilical cord mesenchymal stem cells:
(1) Primary culture (P1) cell number
When one group of cells grew to about 80% confluence in the 8 group of primary cultured cells, the number of P1 generation cells in the 8 group was calculated: the P1 generation cells were digested to prepare a cell suspension, 0.4% (w/v) trypan blue solution was added, and the living cells and the dead cells were counted respectively in three minutes according to the method of white blood cell count of national clinical test procedure (fourth edition), and the total number of living cells was calculated.
(2) Cell culture total number (P5)
After passage of cells to P5, each group of cells was digested to prepare a cell suspension, 0.4% (w/v) trypan blue solution was added, and the living cells and the dead cells were counted respectively in three minutes according to the method of white blood cell count of national clinical test protocol (fourth edition), and the total number of living cells was calculated.
TABLE 3 cell numbers P1 and P5
* P <0.05 vs. the third group; a comparison of # P <0.05 to the seventh group; ﹩ P <0.05 vs. eighth group.
As the results in Table 3 show, the other groups had more cells than the third group for both P1 and P5, and all had statistical differences (P < 0.05). The freezing preservation of the glycerol, uridine and isoleucine on umbilical cord tissues has a certain protection effect. This is mainly because: under the condition of freezing umbilical cord tissue, cells do not have enough nutrition, and substances such as amino acid, fat and the like with certain concentration are added into the frozen stock solution, so that energy can be supplied to vital activities of the cells and the tissues.
As the results in Table 3 show, the number of cells in both P1 and P5 in the fourth group is greater than that in the seventh group, and there are statistical differences (P < 0.05). The uridine has better protective effect on the freezing storage of umbilical tissues than sucrose, creatine and mannitol. This is mainly because: when the umbilical cord tissue is frozen, with the reduction of the temperature, the water inside and outside the cells can be frozen to form ice crystals, the ice crystals can cause mechanical damage, electrolyte elevation, osmotic pressure change, dehydration, pH change, protein denaturation and the like in the cells, the energy supply of the cells to substances such as sugar, amino acid, fat and the like can be limited, and the cells are more likely to turn to use uridine as a nutrient substance.
As the results in Table 3 show, there was no statistical difference in the number of cells of P1 and P5 in the first group (P > 0.05) compared to the eighth group. But the number of P1 cells in the fourth and fifth groups is greater than that of the eighth group P1, with statistical differences; the number of P5 cells in the second, fourth and sixth groups was greater than that of the eighth group P5, with statistical differences. These results demonstrate that the cryopreservation solutions formulated in example 6 are all useful for cryopreserving umbilical cord tissue, and that the second, fourth, fifth, and sixth groups are superior to CryoSure-DEX40 in terms of their effectiveness, with the fourth group being the most effective (Table 3).
Example 7: freezing method effect test
1. Specimen source
The human umbilical cord specimen comes from the term pregnant women of Beijing friendship hospital obstetrics, eliminates fetal malformation, congenital genetic disease and infectious disease history of the women, and signs the informed consent. The donors should be subjected to project examination of human-derived specific viruses (including HIV, HBV, HCV, HTLV, EBV, CMV, etc.), treponema pallidum, glucose-6-phosphate dehydrogenase, and glutamic-pyruvic transaminase, and specific requirements are shown in table 4. Collecting umbilical cord tissue under the aseptic condition of an operating room, washing the umbilical cord tissue for 2-3 times by using normal saline, putting the umbilical cord tissue into a collecting bottle, and transporting the umbilical cord tissue to a laboratory for tissue treatment within 12 hours.
TABLE 4 donor requirements
2. Preparation of umbilical cord samples:
cutting off two ends of the umbilical cord, stroking out blood in umbilical cord blood vessels, dividing the umbilical cord into small sections of about 10cm, soaking for 3min by using a disinfectant, and then soaking and washing for 2-3 times by using a washing liquid until no blood stain exists in the washing liquid. Tearing off two arteries and one vein vessel in the umbilical cord by using elbow forceps, cleaning Wharton's jelly for 2-3 times, and shearing until the Wharton's jelly is 1-2 mm 3 The tissue blocks are divided into three groups according to the average weight, the experiments are divided into three groups, and different operations are respectively carried out:
first group (traditional direct cryopreservation method): a first portion of Wharton's jelly tissue was taken and placed in a freezer containing 2ml of laboratory formulation (the fourth set of freezer in example 6), and about 2-2.5g of tissue per 5ml of freezer tube was placed.
After cooling using a programmed cooling instrument (cooling program: step 1: -10 ℃ C/min to 4.0 ℃ C., step 2: -1.0 ℃ C./min to-8.0 ℃ C., step 3: -25.0 ℃ C./min to-50 ℃ C., step 4: -10.0 ℃ C./min to-14.0 ℃ C., step 5: -1.0 ℃ C./min to-45 ℃ C., step 6: -10.0 ℃ C./min to-90 ℃ C., step 7: end.), the mixture was placed in liquid nitrogen and stored for 3 months.
Taking out the frozen tube, placing in a 37 ℃ constant temperature water bath for 3min, centrifuging at 1500r/min for 5min, removing the frozen solution, and rapidly transferring the tissue suspension to a culture medium (IMDM basal medium+5% platelet lysis)Solution) is centrifuged and washed for 2 to 3 times, the tissue block is transferred to a T25 culture flask and placed in a 37 ℃ and 5 percent CO 2 Culturing in a cell incubator, removing tissue blocks after 5 days, changing fresh medium every 3 days, and culturing at 2×10 when cells grow to about 80% confluence 3 /cm 2 Subculturing is carried out at the density.
Second group (equilibration solution cryopreservation method): a second portion of Wharton's jelly tissue was placed in a freezer containing 4ml of equilibration solution (2% glycerol, 1000. Mu.M uridine, 17.5. 17.5 mM isoleucine, 10% human serum albumin injection, 60% dextran 40 sodium chloride injection), and about 2-2.5g of tissue was placed in each 5ml freezer. Left at room temperature for 45 minutes and then left at 4 ℃ for 15 minutes.
1 volume of cryopreservation agent was added. Freezing agent: 2% glycerol, 1000 mu M uridine, 17.5. 17.5 mM isoleucine, 10% human serum albumin injection, 60% dextran 40 sodium chloride injection, 8% DMSO. After cooling by using a programmed cooling instrument (cooling program: same as the first group), the mixture was put into liquid nitrogen and stored for 3 months.
Taking out the frozen storage tube, placing in a constant-temperature water bath box at 37 ℃ for 3 minutes, centrifuging at 1500r/min for 5 minutes, removing the frozen storage liquid, rapidly transferring the tissue suspension into a sterile centrifuge tube containing a culture medium (IMDM basal medium+5% platelet lysate), centrifuging and washing for 2-3 times, transferring the tissue block into a T25 culture flask, placing in a temperature of 37 ℃ and 5% CO 2 Culturing in a cell incubator, removing tissue blocks after 5 days, changing fresh medium every 3 days, and culturing at 2×10 when cells grow to about 80% confluence 3 /cm 2 Subculturing is carried out at the density.
Third group (equilibration freezing and resuscitation method): a third portion of Wharton's jelly tissue was placed in a freezer containing 4ml of equilibration solution (2% glycerol, 1000. Mu.M uridine, 17.5. 17.5 mM isoleucine, 10% human serum albumin injection, 60% dextran 40 sodium chloride injection), and about 2-2.5g of tissue was placed in each 5ml freezer. Left at room temperature for 45 minutes and then left at 4 ℃ for 15 minutes.
1 volume of cryopreservation agent was added. Freezing agent: 2% glycerol, 1000 mu M uridine, 17.5. 17.5 mM isoleucine, 10% human serum albumin injection, 60% dextran 40 sodium chloride injection, 8% DMSO. After cooling by using a programmed cooling instrument (cooling program: same as the first group), the mixture was put into liquid nitrogen and stored for 3 months.
Taking out the frozen tube, placing in a constant temperature water bath box at 37 ℃ for 3 minutes, centrifuging at 1500r/min for 5 minutes, removing the frozen solution, adding 4ml of balancing solution (2% glycerol, 1000 mu M uridine, 17.5 mM isoleucine, 10% human serum albumin injection, 60% dextran 40 sodium chloride injection), standing at room temperature for 30 minutes, centrifuging at 1500r/min for 5 minutes, and removing the supernatant. Then a mixture of 1 volume of equilibration solution and complete medium (IMDM basal medium+5% platelet lysate) (volume ratio 1:1) was added, and the mixture was left to stand at room temperature for 30 minutes, centrifuged at 1500r/min for 5 minutes, and the supernatant was removed. Rapidly transferring the tissue suspension into a sterile centrifuge tube containing a culture medium (IMDM basal medium+5% platelet lysate), centrifugally washing for 2-3 times, transferring the tissue mass into a T25 culture flask, and placing the T25 culture flask at 37 ℃ and 5% CO 2 Culturing in a cell incubator, removing tissue blocks after 5 days, changing fresh medium every 3 days, and culturing at 2×10 when cells grow to about 80% confluence 3 /cm 2 Subculturing is carried out at the density.
3. Quality detection of umbilical cord mesenchymal stem cells:
quality detection of each quality control point is required to be carried out on UCMSCs after culture is completed, and quality evaluation is carried out on each batch of UCMSCs. Generally includes cell morphology, purity, doubling time, cell viability, safety measures, and the like. UCMSCs quality detection is as follows:
(1) Cell morphology
Taking the 3 rd generation logarithmic growth phase cells, and detecting the adherent cell morphology under a 10-fold optical lens.
(2) Cell viability
Taking cell suspension in the logarithmic growth phase of 3 rd generation, adding 0.4% trypan blue solution, counting living cells and dead cells respectively according to the white blood cell counting method of national clinical test procedure (fourth edition) within three minutes, and calculating the cell viability.
(3) Cell surface markers
Taking 3 rd generation logarithmic growth phase cellsImmunophenotyping, identifying cultured cells as MSCs, digesting with 0.05% pancreatin and collecting cells to make 1×10 6 Each 100. Mu.l of cell suspension was added to each Falcon tube, to which antibodies CD29-APC, CD44-PE, CD90-FITC, CD14-APC, CD19-APC, CD34-PE, CD45-FITC, CD73-PE, CD105-PercP, CD166-PE, HLA-DR-APC, HLA-ABC-FITC were added in advance, and the negative controls were rat IgG1-FITC, igG1-APC, igG1-PE, and IgG 1-PercP. Incubation at 4deg.C for 30min in dark place, PBS washing for 2 times, and flow cytometry detection after resuspension. Data were analyzed using CellQuest Pro.
(4) Identification of cell differentiation-inducing ability
Adipogenic differentiation: taking 3 rd generation logarithmic growth phase cells according to 2×10 4 /cm 2 Density inoculation, adding 2ml of complete culture medium into each well, placing at 37deg.C, 5% CO 2 Culturing in a cell culture box, changing into lipid differentiation complete culture medium A liquid after the cells are completely fused, changing into lipid differentiation induction complete culture medium B liquid after 72 hours, continuously maintaining the lipid differentiation induction complete culture medium B liquid for 7d after 3-5 times of circulation, and changing the liquid for 1 time every 3d during the maintenance period of the B liquid; after differentiation induction was completed, 10% paraformaldehyde was fixed, stained with oil red O, observed under a microscope and photographed.
Osteogenic differentiation: taking 3 rd generation logarithmic growth phase cells according to 2×10 4 /cm 2 Is inoculated in six-hole plates coated with 0.1% gelatin in advance, 2ml of complete medium is added to each hole, and the mixture is placed at 37 ℃ and 5% CO 2 Culturing in an incubator until the cell fusion degree reaches 60% -70%, and then replacing the culture medium with an osteogenic induction differentiation culture medium for 1 time every 3 d; after differentiation induction was completed, 10% paraformaldehyde was fixed, alizarin red stained, observed under a microscope and photographed.
Cartilage-forming induced differentiation: taking 3 rd generation logarithmic growth phase cells, and adjusting cell concentration to 1.6X10 7 /ml. Taking 10 μl of cell suspension, slowly dripping to the middle part of a 6-well plate, culturing for 2h, discarding the growth culture solution and the non-adherent cells, and adding into the cartilage-forming induced differentiation culture medium. After 1 exchange of the liquid every 3d and 21d of induced differentiation, 10% paraformaldehyde was fixed, and the cells were stained with aliskiren blue, observed under a microscope and photographed.
(5) Population doubling time
Taking the 3 rd generation logarithmic growth phase cells, inoculating and culturing for 6 days at a density of 5000 cells/hole (6-hole plate), and calculating the cell doubling time by using the following formula: pdt= (Δt×lg2)/(lgnt—lgn0) formula, PDT: population doubling time; Δt: culturing time; nt: cell number after time t; n0: theoretical initial value of logarithmic growth phase.
(6) Cell clone formation rate assay
Inoculating cells in logarithmic growth phase of 3 rd generation at 100 pieces/hole density into 6-hole plate, slightly rotating to make cells uniform, and placing at 37deg.C and 5% CO 2 Culturing in a cell culture box for 2-3 weeks, frequently observing, stopping culturing when macroscopic cloning clusters appear, discarding supernatant, carefully washing with PBS for 2 times, adding 5ml of 4% paraformaldehyde to fix cells for 15min, removing fixing solution, adding a proper amount of GIMSA to apply staining solution for 30min, then slowly washing the staining solution with flowing water, air-drying, and counting the number of clones of more than 40 cells.
(7) Experiments to inhibit lymphocyte proliferation
Peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) were obtained from healthy donor peripheral blood (30 mL) by Ficoll isolation. Taking UCMSCs in logarithmic growth phase of 3 rd generation, and taking 2×10 4 /cm 2 Inoculating to a 96-well plate for 12h;50 μg/mL mitomycin C for 60 min, 1X 10 added 4 PBMCs, 1 mu g/mL anti-CD3 monoclonal antibody, 1 mu g/mL anti-CD28 monoclonal antibody and 200U/mL IL-2 are added; PBMCs without anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody and IL-2 are taken as blank groups; the PBMCs were co-cultured for 72h, CCK-8 was added, the plates were incubated in an incubator for 2h, and absorbance (OD) at 450nm was measured with a microplate reader. Proliferation rate of PBMCs was calculated as: OD (experimental group)/OD (PBMCs without stimulus). Times.100%. The cell density was adjusted to 1X 10 by taking different groups of PBMCs 6 /mL; adding 10 [ mu ] L of CD25 antibody and 10 [ mu ] L of CD69 antibody respectively; the isotype control tube was added with murine IgG-FITC, igG-PE; incubating at 4 ℃ for 30 min; washing with PBS for 1 time, and centrifuging at 1000 rpm for 5 min; cells were resuspended with 500 μl of PBS and then detected by flow cytometry.
(8) Detection of indoleamine 2, 3-dioxygenase (IDO) Activity
Taking 3 rd generation logarithmic growth phase cells, and adjusting cell concentration to 1×10 5 /cm 2 The method comprises the steps of carrying out a first treatment on the surface of the Treatment with 15ng/mL IFN-gamma or 15ng/mL IFN-gamma+15 ng/mL TNF-alpha for 48h; cell supernatants were collected at 100 μl each and the concentration of immunomodulatory medium IDO was detected using ELISA methods.
(9) Security detection
1) Sterility testing
And (3) inoculating the centrifugal supernatant or the cell culture supernatant to a trypticase soy agar plate culture medium and a Sage dextrose agar plate culture medium, pouring the trypticase soy agar plate culture medium into a 30-35 ℃ incubator for 3-5 days, inverting the Sage dextrose agar plate culture medium into a 20-25 ℃ incubator for 3-5 days, and observing colonies on the plates.
2) Mycoplasma detection
Taking cell suspension in the logarithmic growth phase of the 3 rd generation, and detecting according to the mycoplasma examination method (general rule 3301) in the third part of the 2015 pharmacopoeia of the people's republic of China.
3) Exogenous viral factor detection
Taking cell suspension in the logarithmic growth phase of the 3 rd generation, and detecting exogenous viral factors according to the 2015 edition of the pharmacopoeia of the people's republic of China, the third part (general rule 3302).
4) Endotoxin detection
Taking cell suspension in the logarithmic growth phase of the 3 rd generation, and detecting according to the method of bacterial endotoxin detection (general rule 1143) in 2015 edition of pharmacopoeia of the people's republic of China, third department.
5) Nuclear anomaly rate detection
Cell suspension in logarithmic growth phase of 3 rd generation is taken and detected according to GB 15193.6-2014.
6) Tumorigenicity assay
Taking cell suspension in the logarithmic growth phase of the 3 rd generation, and detecting according to a soft agar clone formation rate detection method, wherein the specific steps are as follows: (1) mixing 1.2% low melting point agarose with 2×cell culture medium at a volume ratio of 1:1 to obtain 0.6% bottom agar, solidifying 1.4ml greenhouse per well in 6-well plate, collectingCells in the logarithmic phase are blown into single cell suspension after being digested by pancreatin, counted and the cell concentration is regulated to 10000 cells/ml; (2) mixing 0.6% low-melting agarose with 2×cell culture medium at a volume ratio of 1:1 to prepare 0.3% upper agar, adding 1ml upper agar and 100ul single cell suspension (about 1000 cells/well) per well, mixing well, and solidifying at room temperature; (3) the 6-well plate was placed at 37℃with 5% CO 2 The cells were cultured in the cell culture chamber for 2 to 3 weeks, and 50 or more cells were counted under a microscope.
(10) Statistical treatment
Statistical analysis using SPSS21.0 software, metering dataThe comparison between groups is shown using one-way analysis of variance. P (P)<A difference of 0.05 is statistically significant.
4. Detection result
(1) Cell morphology observations
Three groups of UCMSCs passed to passage 3, all of which exhibited a uniform long fusiform shape and grew in a vortex as seen in FIG. 1.
(2) Cell phenotype analysis results
Flow cytometry detection showed that UCMSCs from three groups each expressed CD90, CD73, CD105, CD29, CD44, CD166 and HLA-ABC (> 95%) positively, CD14, CD19, CD45, CD34 and HLA-DR (< 2%, table 5) negatively. And there was no statistical difference in immunophenotype expression between the three groups (all P <0.05, table 5)
TABLE 5 results of cell phenotype assays for three groups of UCMSCs
(3) Cell differentiation-inducing ability test results
After 21d of adipogenic induced differentiation of the three groups of UCMSCs, staining with oil red O was performed, and a large amount of lipid deposition, i.e., lipid droplets, was observed under an inverted microscope (FIG. 2).
After 21d of osteogenic differentiation of UCMSCs from the three groups, calcified nodules stained with alizarin red, revealed that densely grown cells were scattered with numerous reddish-orange mineralized nodules (FIG. 2).
After 21d of chondrogenic induced differentiation of the three UCMSCs, blue nodules of different sizes were visualized by staining the cell mass with alisxin blue (FIG. 2, bottom).
(4) Cell viability assay results:
cell viability detection is carried out on UCMSCs in three groups, and the results show that the cell viability among the three groups has no statistical differenceP> 0.05, table 6)
TABLE 6 results of cell viability assays of three groups of UCMSCs
(5) Cell doubling time detection results:
cell doubling time calculation was performed on three groups of UCMSCs, and the results showed that the second group and the third group had less cell doubling time than the first group, and that there was a statistical difference in cell doubling time between the two groups (x)P<0.05, table 7), the third group also had less cell doubling time than the second group, and there was also a statistical difference (#) in cell doubling time between the two groupsP<0.05, table 7). This result indicates the cell doubling capacity: the third group > the second group > the first group.
TABLE 7 results of cell doubling time measurements of UCMSCs in three groups
(6) Clone formation assay test results:
cell clone detection was performed on three groups of UCMSCs, and the results showed that the number of cell clones in the third group and the second group was higher than that in the first group, and that there was a statistical difference in the number of cell clones between the two groups (x)P<0.05, table 8), the third group had a higher number of cell clones than the second group, and there was also a statistical difference (#) in the number of cell clones between the two groupsP<0.05, table 8). This result indicates that the gram of cellsLong Geshu: the third group > the second group > the first group.
TABLE 8 results of cell clone detection of UCMSCs from three groups
(7) Inhibition of lymphocyte proliferation and activation assay results:
the proliferation rate of lymphocytes in the second group and the third group is lower than that in the first group, and the proliferation rate of lymphocytes in the second group and the third group are different statistically from each other (x)P<0.05, table 9), the proliferation rate of lymphocytes of the second group was not statistically different from that of the third groupP> 0.05, table 9). This result indicates the ability of UCMSCs to inhibit lymphocyte proliferation: third group = second group > first group.
The results of the inhibition lymphocyte activation assays performed on three groups of UCMSCs showed that the second and third groups of lymphocytes, CD25 and CD69, had lower expression rates than the first group and were statistically different (x)P<0.05, table 9); the third group of lymphocytes had lower CD25 and CD69 expression rates than the second group, and were statistically different (#)P<0.05, table 6). This result indicates the ability of UCMSCs to inhibit lymphocyte activation: the third group > the second group > the first group.
TABLE 9 results of three groups of UCMSCs inhibition of lymphocyte proliferation and activation experiments
(8) IDO detection results:
IDO is one of the key factors of MSCs for immune regulation, and detection results show that three groups of UCMSCs express IDO at a certain concentration after 48h of treatment with IFN- γ or IFN- γ+tnf- α. The concentration of IDO in both the third and second groups was higher than that in the first group, with statistical differences (< 0.05 for P, table 10); and the concentration of IDO in the third group was also higher than in the second group, with statistical differences (#p <0.05, table 10); these results indicate that UCMSCs have immunomodulatory functions: the third group > the second group > the first group.
TABLE 10 detection results of IDO Activity in three groups (pg/ml)
(9) Safety detection result:
the results of the safety detection of the UCMSCs in the three groups show that the UCMSCs have no tumorigenicity and abnormal karyotype under the tissue block freezing storage mode, have very high biological safety, and meet the microbiological safety requirements of clinical application.
TABLE 11 safety Performance test results
In a word, the freezing effect of the method for freezing the umbilical cord tissue by the balancing liquid is better than that of the traditional direct freezing method, and the balancing liquid freezing and recovering method is also better than that of the balancing liquid freezing.
Visible. The use of a equilibration solution pre-treatment prior to cryopreservation and/or during resuscitation can reduce damage to MSCs from freezing, improve yield and efficacy of cell resuscitation, such as increasing cell doubling capacity, enhancing ability to inhibit lymphocyte proliferation, improving immune regulation, etc.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (6)

1. The freezing preservation protective agent is characterized by comprising a balancing liquid and a freezing preservation liquid;
the balance liquid consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection;
the frozen stock solution consists of the following components in the final concentration: 5-10% of DMSO (dimethyl sulfoxide), 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection;
placing the umbilical cord tissue blocks in balance liquid for standing, and then placing the umbilical cord tissue blocks in frozen stock solution for cooling and preserving.
2. The freezing preservation protective agent is characterized by comprising a balancing liquid and a freezing preservation liquid;
the balance liquid consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection;
the frozen stock solution consists of the following components in the final concentration: 0.5-5% of glycerin, 100-2000 mu M of uridine, 2.5-25 mM of isoleucine, 5-10% of DMSO, 5-20% of human serum albumin injection and 30-80% of dextran 40 sodium chloride injection;
placing the umbilical cord tissue blocks in balance liquid for standing, and then placing the umbilical cord tissue blocks in frozen stock solution for cooling and preserving.
3. A kit for umbilical cord tissue, comprising: a cryoprotectant as claimed in claim 1 or claim 2.
4. A method for cryopreserving umbilical cord tissue, comprising: cryopreserving the umbilical cord tissue mass using the cryopreservation protectant of claim 1 or 2: placing the umbilical cord tissue blocks in balance liquid for standing, and then placing the umbilical cord tissue blocks in frozen stock solution for cooling and preserving.
5. A method for constructing a cell tissue library of umbilical cord tissue, comprising: the method for freezing and preserving umbilical cord tissue blocks according to claim 4, and resuscitating the frozen umbilical cord tissue blocks.
6. The method for constructing a tissue bank of umbilical cord tissue as claimed in claim 5, wherein the step of resuscitating the cryopreserved umbilical cord tissue mass comprises:
thawing and dissolving the frozen umbilical cord tissue blocks, and centrifuging to remove supernatant; adding the balancing solution in claim 1 into the centrifuged product, standing for 10-40min, centrifuging to remove supernatant, adding mixture of balancing solution and complete culture medium, standing for 10-40min, centrifuging to remove supernatant, culturing cells in placenta tissue mass, and amplifying to obtain cells.
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