CN117106706A - Method for separating mesenchymal stem cells from placenta and corresponding preservation method of tissue derived from mesenchymal stem cells - Google Patents
Method for separating mesenchymal stem cells from placenta and corresponding preservation method of tissue derived from mesenchymal stem cells Download PDFInfo
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- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- 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
- C12N2509/10—Mechanical dissociation
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Abstract
The invention discloses a method for separating mesenchymal stem cells from placenta and a corresponding preservation method of tissue derived from the mesenchymal stem cells; the method comprises the following steps: adding physiological solution containing collagenase I into placenta tissue, and digesting for 1-3 hr; solid-liquid separation, collecting tissue blocks which are not completely digested; and inoculating the tissue blocks which are not completely digested into a culture medium for wall-attaching culture to obtain mesenchymal stem cells. The method for separating the mesenchymal stem cells from the placenta adopts collagenase digestion to gently decompose the placenta tissue into tiny tissue blocks with the size of about 0.5mm, so that the damage and the influence of the tiny tissues on the cells due to mechanical cutting are avoided. The tissue block is compact, and can be quickly settled and attached after inoculation, wherein MSC can be quickly attached and grown; meanwhile, the tissue block can be preserved for a long time in liquid nitrogen by adopting the preservation method of the scheme of the invention, and can be used as a source tissue of MSC seed cells for resuscitation at any time.
Description
Technical Field
The invention belongs to the field of stem cells, and particularly relates to a method for separating mesenchymal stem cells from placenta and a corresponding preservation method of tissue derived from the mesenchymal stem cells.
Background
Mesenchymal stem cells (mesenchymalstem cells, or mesenchymal stromal cells, MSC) are adult stem cells with multidirectional differentiation capability, widely exist in various tissues of a body, can differentiate into various tissue cells, and participate in forming a tissue regeneration microenvironment by secreting a large amount of cytokines, so that tissue injury repair is promoted, and body immune response is regulated. The MSC can be derived from umbilical cord, placenta, bone marrow, fat, etc. Placenta belongs to a 'medical waste' tissue, meets the ethical requirement, has the advantages of large single tissue quantity, young tissue, rich MSC content, rich tissue sources and the like, and is an ideal tissue source for developing MSC scale preparation for patent medicine research.
The method for separating MSC from placenta mainly comprises enzymolysis method and tissue adherence method. The enzymolysis method is to carry out enzyme digestion on small tissue blocks to obtain single cell suspension, and separate MSC from the single cell suspension; the method has the defects that dense tissues are difficult to be subjected to enzymolysis, and the long-time digestion has great damage to cells, so that the survival and biological characteristics of the cells are affected; excessive mechanical cutting of tissue can also affect the survival and biological properties of cells within the tissue. Therefore, the tissue is usually subjected to a certain enzymolysis, a part of cells are obtained, and the rest of the tissue is discarded. Tissue adherence is an important method for separating cells capable of adhering to and growing in tissues, provided that the density of the tissues is higher than that of the culture medium, and the tissues can be adhered to the surface of a culture dish by gravity; MSCs are characterized by their strong adherent capacity and their ability to grow adherent, and therefore, this method is widely used for isolating MSCs from umbilical cord tissue (which has a specific gravity greater than that of the culture medium). But the success rate of separating MSC by placenta tissue adherence method is low, mainly because the placenta tissue has low overall density (less than water) and floats in the culture medium. Placenta tissue is composed of irregular tree-like dense tissue and gaps (blood is filled in physiological state) therebetween, and the dense tissue is difficult to separate by mechanical cutting. The small tissue formed by directly shearing placenta tissue has flocculent part after washing with normal saline, and is easy to float after being inoculated into a culture bottle (or cell factory) containing a culture medium, so that MSC in the tissue cannot carry out adherent growth, and MSC separation is difficult to realize.
Tissue cryopreservation is an important method to preserve the original state of cells and the integrity of gene expression. Because the current tissue freezing and preserving procedure is complex, the commercial tissue freezing and preserving solution is expensive, and the effective tissue freezing and preserving volume is small (at 1 mm) 3 In) most commercial tissue cryopreservation is actually the preservation of cells obtained after tissue lysis or cells obtained after further culturing of cells within the tissue. About 500g of human placenta tissue, it is too expensive to freeze whole tissue, and it is a challenge to selectively preserve stem cell-derived tissue to the maximum extent.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. For this purpose, the present invention proposes a method for isolating mesenchymal stem cells from placenta.
The invention also provides the mesenchymal stem cells separated by the method for separating the mesenchymal stem cells from the placenta.
The invention also provides application of the method for separating mesenchymal stem cells from placenta.
The invention also provides a preservation method of the mesenchymal stem cells.
According to one aspect of the present invention, there is provided a method of isolating mesenchymal stem cells from a placenta, the method comprising the steps of: adding physiological solution containing collagenase I into placenta tissue, and digesting for 1-3 hr; solid-liquid separation, collecting tissue blocks which are not completely digested; and inoculating the tissue blocks which are not completely digested into a culture medium for wall-attaching culture to obtain mesenchymal stem cells.
In some embodiments of the invention, the method further comprises the step of pre-treating the placental tissue, the step comprising: washing placenta tissue, and dividing into small pieces of placenta tissue with square size of 2-3 cm; and (3) cleaning the placenta tissue small block, and then dividing the placenta tissue small block into small tissue blocks with square 2-3 mm.
In some embodiments of the invention, the washing is performed with physiological saline containing 10-30 μg/mL gentamicin sulfate.
In some embodiments of the invention, the physiological saline has a temperature of 2 to 10 ℃.
In some embodiments of the invention, the number of washes is 2 to 5.
In some embodiments of the invention, the dividing is by using scissors.
In some embodiments of the present invention, the method further comprises the step of adding a DMEM solution to the small tissue mass, performing solid-liquid separation, and collecting the solid phase.
In some embodiments of the invention, the mass to volume ratio of small tissue pieces to DMEM solution is (1-2): 1-4 (g/mL).
In some embodiments of the invention, the DMEM solution contains gentamicin sulfate at a final concentration of 10-30 μg/mL.
In some embodiments of the invention, the physiological solution comprises DMEM.
In some embodiments of the invention, the collagen I enzyme is present in the physiological solution in an amount of 0.1 to 5% by mass.
In some embodiments of the invention, the collagen I enzyme is present in the physiological solution in a mass percentage of 0.1 to 1%.
In some embodiments of the invention, the collagen I enzyme is present in the physiological solution at a mass percentage of 0.5%.
In some embodiments of the invention, the physiological solution further comprises 0.000001-0.00001% calcium chloride.
In some embodiments of the invention, the size of the undigested complete tissue mass is (200-800) × (200-800) μm.
In some embodiments of the invention, the temperature of the digestion is between 32 and 40 ℃.
In some embodiments of the invention, the step of solid-liquid centrifugation is as follows: adding physiological solution containing collagenase I into placenta tissue, digesting to obtain digestive juice, sieving with 20-40 mesh cell sieve, and collecting filtrate; centrifuging the filtrate, and collecting precipitate; the pellet was resuspended in DMEM solution and then passed through a 70-90 mesh cell sieve.
In some embodiments of the invention, further comprising washing the 70-90 mesh cell sieve described above with DMEM solution; collecting the flushing liquid, carrying out solid-liquid separation on the flushing liquid, and collecting a solid phase to obtain the micro tissue.
In some embodiments of the invention, the solid-liquid separation is performed by centrifugation at 200-500g for 3-7min at 2-6deg.C.
In some embodiments of the invention, the DMEM solution is at a temperature of 2-10 ℃.
In some embodiments of the invention, the medium comprises MSC complete medium.
In some embodiments of the invention, the medium contains 5-12% fetal bovine serum at a final concentration.
In some embodiments of the invention, the mass to volume ratio of incompletely digested tissue pieces to medium added is (0.05-2): (10-14) g/mL.
In some embodiments of the invention, the culture conditions of the expansion culture are 35-38deg.C, 4-6% CO 2 。
In some embodiments of the invention, the liquid change method of the expansion culture is to culture for 70-74h, the liquid is changed once, and the liquid change culture medium is a culture medium at 35-40 ℃.
In some embodiments of the invention, the time of the expansion culture is 3-10d.
In some embodiments of the invention, the method of collecting the mesenchymal cells is as follows: removing the supernatant from the culture medium obtained by the expansion culture, adopting a trypsin solution to digest for 4-5min, stopping digestion, and collecting cell suspension; filtering the cell suspension, and collecting the filtrate; and (3) carrying out solid-liquid separation on the filtrate, and collecting a solid phase, namely the mesenchymal stem cells.
In some embodiments of the invention, the trypsin comprises 0.01-0.08% by weight of the trypsin solution.
In some embodiments of the invention, the trypsin solution further comprises EDTA at a final concentration of 0.3-0.7 mM.
In some embodiments of the invention, the terminating digestion employs DMEM containing 8-12% FBS.
In some embodiments of the invention, the filtration is performed using a cell screen having a pore size of 30-50 μm.
In some embodiments of the invention, the solid-liquid separation is performed by centrifugation at 200-500g for 3-7min at 20-30deg.C.
In some embodiments of the invention, the method further comprises the step of passaging the resulting mesenchymal stem cells, said passaging employing 8000-10000 MSCs/cm 2 Is inoculated at a density of the culture medium for subculture.
According to a second aspect of the present invention, mesenchymal stem cells isolated by the above method are provided.
In some embodiments of the invention, the positive rate of at least one of the markers CD90, CD105 and CD73 in the mesenchymal stem cells is greater than 98%.
In some embodiments of the invention, the positive rate of at least one of the markers CD34, CD11b, CD19, CD45 and HLA-DR in the mesenchymal stem cells is less than 1%.
According to a third aspect of the present invention, there is provided the use of the above method in the preparation of mesenchymal stem cells.
According to a fourth aspect of the present invention, there is provided a preservation method of tissue derived from mesenchymal stem cells, comprising the steps of: after the frozen stock solution is added into the tissue block which is not completely digested and prepared by the method, the procedure is carried out, and then the liquid nitrogen is stored.
In some embodiments of the invention, the programmed cooling comprises the steps of: and after the first gradient cooling to-48 to-52 ℃, heating to-12 to-15 ℃, and the second gradient cooling to-88 to-92 ℃, transferring to liquid nitrogen for freezing storage.
In some embodiments of the invention, the first gradient cooling comprises the steps of: mixing the tissue blocks which are not completely digested with the frozen stock solution, regulating the temperature to be 2-6 ℃, and cooling to-5 to-7 ℃ at the speed of 0.5-2 ℃/min; then cooling to-48 to-52 ℃ at a speed of 22-28 ℃/min.
In some embodiments of the invention, the temperature is raised to-12 to-15 ℃ rapidly at 22 to 28 ℃/min after the first gradient is cooled to-48 to-52 ℃.
In some embodiments of the present invention, the second gradient cooling means that after the temperature is raised to-12 to-15 ℃, the temperature is lowered to-40 to-50 ℃ at 0.5 to 3 ℃/min, and then the temperature is lowered to-80 to-100 ℃ at 8 to 12 ℃/min.
In some embodiments of the invention, the cryopreservation solution is serum containing 7-12% dimethyl sulfoxide (DMSO).
In some embodiments of the invention, the temperature of the frozen stock solution is 2 to 6 ℃.
In some embodiments of the invention, the serum comprises fetal bovine serum.
According to some embodiments of the invention, at least the following benefits are provided: the method for separating the mesenchymal stem cells from the placenta adopts collagenase digestion to gently decompose the placenta tissue into tiny tissue blocks with the size of about 0.5mm, so as to avoid the damage and influence of the tiny tissue mechanically cut into cells. The tissue block is denser, the specific gravity is greater than that of an MSC culture medium, the tissue block is quickly settled and attached after inoculation, and MSC in the tissue block quickly attached and grows; meanwhile, the tissue block can be preserved for a long time in liquid nitrogen by adopting the preservation method of the scheme of the invention, and can be used as a source tissue of MSC seed cells for resuscitation at any time.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing the results of detecting the growth of peripheral cells of a microstructure observed under a microscope 3 days after inoculation of a micro tissue mass in example 1 of the present invention;
FIG. 2 is a graph showing the results of cell confluence detection after 7 days of culture by the micro tissue adherence method and the simple tissue adherence method after enzymolysis in example 1 of the present invention;
FIG. 3 is a graph showing the results of cell productivity measurement after 7 days of culture by the micro tissue adherence method and the simple tissue adherence method after enzymolysis in example 1 of the present invention;
FIG. 4 is a graph showing the results of flow cytometry analysis of the expression of the 1 st generation (P1 st generation) surface markers of cell culture obtained by the micro-tissue adherence method after enzymatic hydrolysis in the test example of the present invention;
FIG. 5 is a graph showing the expression of the surface markers of cultured 6 th (P6 th) MSC obtained by flow cytometry analysis of the micro tissue adherence method after enzymatic hydrolysis in the test example of the present invention;
FIG. 6 is a graph showing the results of differential staining of the three lines of MSC obtained by the method of attaching micro tissue to the substrate after enzymolysis in the test example of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1A method of isolating mesenchymal Stem cells from placenta
The embodiment provides a method for separating mesenchymal stem cells from placenta, which comprises the following specific processes:
(1) The maternal surface tissue of the placenta was cut (about 1/2 placenta thickness) and the remaining placental fetal surface tissue was washed 3 times with pre-chilled saline containing gentamicin sulfate at a final concentration of 20 μg/mL.
(2) The placenta tissue is cut into small pieces with the square of 2-3cm by scissors, and then washed 3 times by normal saline containing gentamicin sulfate with the final concentration of 20 mug/mL.
(3) The tissue was added to pre-chilled low-sugar DMEM (Dulbecco's Modified Eagle Medium, corning) containing gentamicin sulfate at a final concentration of 20. Mu.g/mL or Phosphate Buffered Saline (PBS) to soak the tissue and stored at 2-8deg.C.
(4) 2-3 pieces of tissue are clamped from a centrifugal bottle containing the tissue by using sterile forceps, the tissue is placed in a 10cm culture dish, the culture dish is placed on an ice box, and the sample tissue is rapidly sheared into small tissue pieces with the square of 2-3mm by using sterile surgical scissors.
(5) The minced tissue collection was transferred to a 250mL centrifuge bottle containing 20 μg/mL gentamycin sulfate low-sugar DMEM or PBS using a sterile stainless steel drug spoon.
(6) The tissue was homogenized in a ratio of 50g tissue/100 mL DMEM, centrifuged at 300g at 4℃for 3min, and the supernatant was discarded.
(7) Collagenase digestion: 50g of the minced tissue obtained in the step (6) was placed in a 250mL centrifuge bottle, 75mL of low-sugar DMEM or PBS containing 0.5% collagen I enzyme (purchased from Sigma) and 0.000005% calcium chloride was added, mixed uniformly, incubated at 37℃for 120 minutes, and mixed uniformly by shaking every 10 minutes for 1 time. Mixing the digestate uniformly, sieving with a 30-mesh sterile stainless steel sieve, and removing undigested tissues; the filtrate was centrifuged at 530g at 4℃for 10min, the supernatant was discarded, the pellet was resuspended in 200mL cold DMEM, and the mixture was stirred well and sieved through a 80 mesh stainless steel screen.
(8) Collecting small tissue blocks: turning over the cell sieve, placing the cell sieve on a sterile beaker, flushing with precooled low-sugar DMEM or PBS, collecting tiny tissues on the sieve, collecting flushing liquid containing the tiny tissues, centrifuging for 5min at 4 ℃, discarding the supernatant, and dividing the sediment part into tiny tissue blocks with the size of 200-800 multiplied by 200-800 mu m, and most of the tiny tissue blocks are about 500 multiplied by 500 mu m.
(9) Micro tissue mass inoculation: the micro tissue blocks are resuspended with MSC complete medium in an amount that is usual for MSC culture without reducing the amount of medium to prevent floating of the tissue blocks. If the micro tissue block is mixed with 12mL of MSC complete medium (low sugar DMEM containing 10% Fetal Bovine Serum (FBS)) according to the proportion of 100mg (μl), inoculated into a T75 culture flask, placed in an incubator at 37 ℃ and 5% CO 2 Culturing for 72h without shaking. Wherein fresh medium was changed every 72 hours, the flask was gently tilted, medium was slowly aspirated off, and then MSC complete medium pre-warmed at 37 ℃ was slowly added.
(10) Harvesting the cells: from day 3, the cells were observed daily using a microscope. On day 3 of culture, the results of microscopic observation are shown in FIG. 1, and it can be seen from the figure that almost all of the microscopic tissue mass had cells grown around, after which the cells gradually grew around. After culturing for 7 days, the results of observation under a microscope are shown in a diagram in FIG. 2, and it can be seen from the diagram that a cell band of about 150 μm wide is formed around a tissue mass, and cells between partial tissue masses are nearly confluent, so that the cells can be collected. Method for collecting cells: the culture supernatant was aspirated, the flask was washed 2 times with Phosphate Buffered Saline (PBS), 2mL of pre-warmed 0.05% trypsin containing 0.53mM EDTA was added, and incubated in an incubator at 37℃for 4-5min. The flask was gently tapped, 4mL of DMEM containing 10% FBS was added to terminate digestion, the cell suspension was collected by pipetting several times, the cell suspension was filtered with a cell filter screen having a pore size of 40 μm, the filtrate was collected, centrifuged for 5min,400g, and the supernatant was discarded after centrifugation, and the mesenchymal stem cells were collected. Cell pellet was resuspended in DMEM containing 10% FBS and cells were counted using Cellcounter, and 120 ten thousand cells were harvested per T75 flask.
(11) Subculture: according to 9000 MSCs/cm 2 And (3) subculturing the mesenchymal stem cells obtained in the step (10) of density inoculation.
Experimental results show that MSC is obtained by using an enzymolysis micro-tissue adherence method, the cell yield is high, tissue blocks can be quickly adhered to a culture dish after inoculation, and cells grow out around almost all micro-tissues when the culture is performed for 3 days; after 7 days of culture, the cell confluency had reached passable density, and 122 ten thousand cells were harvested per T75 flask (as shown in fig. 3).
Example 2
The embodiment provides a method for freezing and storing micro tissue blocks prepared in the embodiment 1, which comprises the following specific processes:
1. freezing and preserving micro tissues: 0.5g (mL) of the micro tissue block prepared in example 1 was taken, 4.5mL of FBS (precooled) containing 10% dimethyl sulfoxide (DMSO) was added, and the mixture was rapidly and uniformly mixed, and the mixture was dispensed into 5 freezing tubes at a concentration of 1 mL/tube.
2. Rapidly transferring the cell cryopreservation tube into a program cooling instrument (started and waiting at 4.0 ℃), and carrying out gradient cooling, wherein the cryopreservation program is as follows:
Step 1：Wait at 4℃；
Step 2：1℃/min to-6℃；
Step 3：25℃/min to-50℃；
Step 4：25℃/min to-14℃；
Step 5：1℃/min to-45℃；
Step 6：10℃/min to-90℃；
Step 7：Hold-90℃for 5min；
step 8: transfer cells to liquid nitrogen tank for preservation.
Comparative example 1
The comparative example provides a method for separating mesenchymal stem cells from placenta, namely placenta tissues are directly cultured by a tissue adherence method without enzyme digestion, and the growth of the cells is observed. The method specifically comprises the following steps:
1) The method of washing, cutting and shearing placenta tissue was the same as in steps (1) to (6) of example 1.
2) Inoculating: the inoculation density was one T75 flask per 0.1g of tissue. The method comprises mixing 100mg of tissue with 12mL of low-sugar DMEM containing 10% FBS, inoculating into T75 culture flask, placing into incubator at 37deg.C and 5% CO 2 Culturing under the condition, continuously culturing for 72 hours, and not shaking the culture bottle during the culture period.
3) Liquid replacement: fresh medium was changed every 72 hours by gently tilting the flask, slowly pipetting the medium, and then slowly adding 37℃pre-warmed MSC complete medium.
4) Observing the growth condition of cells: cell growth was observed daily starting on day 3. On day 3 of culture, about 10% of the tissue pieces had small numbers of cells grown out, significantly less than the enzyme digested microscopic tissue pieces. On day 3 of culture, the tissue mass with cells growing thereon continued to adhere, and the cells became more numerous (shown in panel B of fig. 2). The tissue mass growing without cells floats and necrosis.
5) Harvesting the cells: after 7 days of incubation, the culture supernatant was discarded, the flask was washed 2 times with phosphate buffer PBS, and 2mL of 0.05% pancreatin containing 0.53mM EDTA was added and incubated in an incubator at 37℃for 4-5min. The flask was gently tapped, 4mL of DMEM containing 10% FBS was added to terminate digestion, the flask was pipetted several times, and the flask was filtered through a 40 μm cell sieve, and the filtrate was centrifuged for 5min at 400g at room temperature.
6) Cell count: after centrifugation, the supernatant was discarded, the cells were resuspended, and the cells were counted using a Cellcounter, and 3.3 ten thousand cells were harvested per T75 flask (as shown in fig. 3).
Experimental results show that the isolated MSC yield of placenta tissue by a direct adherence method is very low, most tissue blocks float after inoculation and cannot be firmly attached to a culture dish, and no cells grow out. Only a few tissues had cells growing out, but the time was late, the number of harvested cells was small when cultured for 7 days, and the number of harvested cells per T75 flask was only 1/37 of that of the micro-tissue adhesion method of the present invention.
Comparative example 2
The comparative example provides a method for isolating mesenchymal stem cells from placenta, specifically comprising the following steps:
after the placenta tissue is digested by enzyme, medium-sized tissue is cultured by an adherence method, and the growth of cells is observed.
1) The method of washing, cutting and shearing placenta tissue was the same as in steps (1) to (6) of example 1.
2) Collagenase digestion: 50g of the sheared tissue was placed in a 250mL centrifuge bottle, 75mL of low sugar DMEM containing 0.5% collagen I enzyme (Sigma) and 0.000005% calcium chloride was added, mixed well, incubated at 37℃for 120 minutes, and mixed well with shaking 1 time every 10 minutes. The digestate was mixed well and sieved through a 30 mesh sterile stainless steel screen.
3) Collecting tissue blocks: turning over the cell sieve, placing on a sterile beaker, washing with pre-cooled DMEM, collecting placenta tissue on the sieve, collecting washing liquid of placenta tissue, centrifuging at 4deg.C for 5min at 300g, discarding supernatant, and collecting the precipitate as placenta tissue block with medium size of about 1500-2500 μm.
4) Inoculating: the inoculation density was one T75 flask per 100mg of tissue. The method comprises mixing 100mg of tissue with 12mL of low-sugar DMEM containing 10% FBS, inoculating into T75 culture flask, placing into incubator at 37deg.C and 5% CO 2 Culturing under the condition, continuously culturing for 72 hours, and not shaking the culture bottle during the culture period.
5) Liquid replacement: fresh medium was changed every 72 hours by gently tilting the flask, slowly pipetting the medium, and then slowly adding 37℃pre-warmed MSC complete medium.
6) Observing the growth condition of cells: cell growth was observed daily starting on day 3. On day 3 of culture, about 10% of the tissue pieces had small numbers of cells grown out, significantly less than the enzyme digested microscopic tissue pieces. On day 3 of culture, the tissue mass with cells growing thereon continued to adhere, and the cells became more numerous (as shown in panel C of FIG. 2). The tissue mass growing without cells floats and necrosis.
7) Harvesting the cells: after 7 days of incubation, the culture supernatant was discarded, the flask was washed 2 times with phosphate buffer PBS, and 2mL of 0.05% pancreatin containing 0.53mM EDTA was added and incubated in an incubator at 37℃for 4-5min. The flask was gently tapped, 4mL of DMEM containing 10% FBS was added to terminate digestion, the flask was pipetted several times, and the flask was filtered through a 40 μm cell sieve, and the filtrate was centrifuged for 5min at 400g at room temperature.
8) Cell count: after centrifugation, the supernatant was discarded, the cells were resuspended, and the cells were counted using a Cellcounter. The results of the counts are shown in FIG. 3, 6.8 ten thousand cells were harvested per T75 flask. Experimental results show that the MSC yield of the medium-size tissue block adherence method is very low, most tissue blocks float after inoculation and cannot be firmly attached to a culture dish, and no cells grow out. Only a few tissues had cells growing out, but the time was late, the number of harvested cells was small when cultured for 7 days, and the number of harvested cells per T75 flask was only 1/18 of that of the micro-tissue adhesion method of the present invention.
Test case
1. MSC surface marker detection
The cells obtained in example 1 were subcultured to P6 passages. Cells were harvested at the P1 and P6 passages, respectively, and expression of MSC surface markers was detected using a flow cytometer. The detection method comprises the following steps:
(1) Removing culture supernatant, washing the culture flask with PBS for 2 times, adding a proper amount of 0.05% pancreatin containing 0.53mM EDTA, digesting for 2-3 min at 37 ℃, blowing into single cells by a pipette, adding a proper amount of DMEM culture medium containing 10% FBS, stopping digestion, centrifuging, and washing with PBS once;
(2) Cells were resuspended in PBS containing 1% bovine serum albumin, split into different EP tubes of 10-20 tens of thousands of cells per 100. Mu.L, and incubated on ice for 30min with the addition of APC, PE or FITC labeled CD90, CD105, CD73, CD34, CD11b, CD19, CD45, HLA-DR and their corresponding isotype control IgG 2. Mu.L;
(3) Centrifuging at 400g and 4deg.C for 5min;
(4) The supernatant was discarded, and after washing the cells with PBS, they were examined by flow cytometry.
As shown in FIGS. 4 and 5, it can be seen that the positive rate of the markers CD90, CD105 and CD73, which are characteristic of MSC, is greater than 98% and the positive rate of CD34, CD11b, CD19, CD45 and HLA-DR is less than 1% after the cells obtained by the micro tissue adherence method are cultured at the initial stage (P1) and continuously until the cells are cultured to P6. The results show that the cells obtained by the enzymolysis method and the tissue adherence method accord with the expression characteristics of the MSC surface markers.
2. Three-line differentiation study
The cells obtained in example 1 were subcultured to P6 passages. Cells were seeded into 12-well plates at passage P6 for three-line differentiation studies.
The experimental method comprises the following steps:
(1) MSC was inoculated in 12-well plates at an inoculum size of 13 ten thousand per well, and the cells were divided into 4 groups of 3 wells each, which were a control group (without induction culture), an osteogenic differentiation group, a chondrogenic differentiation group, and a adipogenic differentiation group, respectively.
(2) After MSCs were cultured in low-sugar DMEM containing 10% FBS to 80% confluency, 1mL of adipogenic differentiation medium (Biological Industries), osteogenic differentiation medium (Biological Industries) and chondrogenic differentiation medium (Biological Industries) were added to each of the three differentiation groups, respectively, and the control group continued to be cultured with the MSC medium.
(3) The corresponding fresh medium was replaced every 72 h.
(4) At the time of 14 days of induction culture, the induced differentiation groups were respectively subjected to adipogenic, osteogenic, and chondrogenic staining. 1 well in the control group served as the uninduced control for corresponding staining.
The fat dyeing method comprises the following steps:
(1) Preparation of Oled-O mother liquor: 0.35g Oli red-O (Sigma 00625) was weighed out and dissolved with 100mL isopropyl alcohol (> 99.5%); filtered through a 0.22 μm filter and stored at 2-8deg.C in the dark.
(2) Preparing Oled-O dyeing working solution: taking Oled-O mother liquor and distilled water according to the following ratio of 3:2, standing for 20min, and filtering with 0.22 μm filter for use (for 2-3 hr).
(3) The 12-well plate medium was aspirated and gently rinsed 2 times with 1.5mL PBS.
(4) Fixing: the PBS was removed by blotting, 1.5mL of 4% paraformaldehyde (PFA, sigma) was added to each well, and the wells were fixed at room temperature for 60min.
(5) PFA was blotted off and washed with 1.5 mL/well 60% isopropyl alcohol for 3min.
(6) The isopropanol was removed by suction, 1.5mL of Oled-O staining solution was added thereto, and the mixture was allowed to stand at room temperature for 30 minutes.
(7) The dye was removed by blotting and washing 2 times with 1.5mL distilled water per well to wash off excess dye.
(8) 1mL of distilled water was added, and the resultant was observed under a microscope to give a lipid-forming effect, and photographed.
The osteogenic staining method is as follows:
an osteogenic ARS staining kit (Vivacell C37C 0-0150) was used.
(1) The medium was aspirated and washed once with 1.5mL PBS;
(2) Fixing: the PBS was removed by blotting, 1.5. 1.5mL Fixation solution (C37C 10020) was added to each well to homogenize the bottom surface and fix it at room temperature for 60min;
(3) The Fixation solution wells were blotted, 1.5mL of Wash I (C37C 20050) was added to each well, and the cells were gently rinsed 3 times;
(4) Sucking Wash I, adding 1.5. 1.5mL Staining Solution (C37C 30020) into each hole, soaking the bottom surface uniformly, and standing at room temperature for 60min;
(5) The Staining Solution wells were blotted, 1.5mL Wash II (C37C 40050) added per well, and the cells gently rinsed 4 times;
(6) Wash II was pipetted off, 1mL Inspection Solution (C37C 50010) was added to each well, and the wells were observed under a microscope and photographed.
The method for staining the cartilage comprises the following steps:
(1) 1% Alcian Blue dyeing working solution was prepared: weighing 0.2g Alcian Blue 8GX (Sigma), dissolving with 20ml of 0.1N HCl, filtering with 0.22 μm filter, and storing in refrigerator at 2-8deg.C in dark place;
(2) The medium was aspirated and gently washed once with 1.5mL PBS;
(3) Fixing: the PBS was removed by blotting, 1.5mL of 4% PFA was added to each well, and the mixture was fixed at room temperature for 60min;
(4) 4% PFA was blotted off and each well was washed 2 times with 1.5mL distilled water;
(5) Distilled water is sucked off, 1.5mL of 1% Alcian Blue dyeing working solution is added, and the solution is kept away from light at room temperature for overnight;
(6) The dye liquor was removed by suction, and 1mL of 0.1N HCl was added to each well to wash 3 times;
(7) HCl was removed by blotting, 1mL of distilled water was added to each well, and the mixture was observed under a microscope and photographed.
The results of the three-line differentiation are shown in FIG. 6, and it can be seen from the graph that after MSC adipogenesis induction, the staining results show that the cells contain orange-colored vesicles (fat droplets), demonstrating that the cells develop adipogenic differentiation. The osteogenic differentiation staining results showed that after MSCs were cultured by osteogenesis, the cells produced calcium nodules, indicating that osteogenic differentiation occurred. The results of chondrogenic differentiation staining showed that MSCs were cultured by chondrogenic induction and cells stained positive for Alcian Blue, indicating that chondrogenic differentiation occurred. Cells of the control group were negative for the staining described above.
3. Micro-tissue resuscitation and MSC separation
The frozen micro-tissue of example 2 was resuscitated and MSC isolation verified as follows:
(1) Taking out 2 frozen micro tissues from the liquid nitrogen tank after the micro tissues are frozen for 6 months, rapidly putting the micro tissues into a cell transfer bottle containing liquid nitrogen, and transferring the micro tissues to a cell culture room;
(2) Respectively adding 10mL of mesenchymal stem cell complete medium which has been rewarmed to 37 ℃ into 2 10mL centrifuge tubes in a biosafety cabinet;
(3) Taking out the cell freezing tube from the transfer bottle by using tweezers, vertically placing the cell freezing tube in a37 ℃ water bath kettle by using a buoy to clamp the cell freezing tube, immersing the lower 2/3 of the tube body into water, slightly shaking until the ice in the cell freezing tube is melted into liquid, and immediately taking out the cell freezing tube from the water bath kettle;
(4) Transferring all the micro-tissue suspension into a centrifuge tube containing a rewarmed mesenchymal stem cell complete culture medium by using a pipette in a biosafety cabinet, and lightly blowing and uniformly mixing;
(5) And (3) centrifuging: centrifuging the micro-tissue suspension at 400g and room temperature for 5min;
(6) The supernatant was aspirated, 12mL of MSC complete medium resuspended cells containing 10% FBS were added to each centrifuge tube, inoculated into T75 flasks, placed at 37℃and 5% CO, respectively 2 Culturing in an incubator; liquid replacement: fresh medium was changed every 72 hours by gently tilting the flask, slowly pipetting the medium, and then slowly adding 37℃pre-warmed MSC complete medium.
The results were as follows: on day 3 of culture, cells grew around almost all the pieces of micro tissue under the microscope, and the situation was similar to that of fresh micro tissue cultured, and thereafter the cell growth was similar to that of fresh micro tissue, and by day 7 of culture, the cell confluency reached passable level. After 4-5min of trypsin/EDTA digestion at 37℃and termination of MSC complete medium, cell suspension was collected, filtered through a cell filter with a pore size of 40. Mu.m, filtrate was collected, centrifuged for 5min,400g, at normal temperature, and the supernatant was discarded after centrifugation. Cells were resuspended in MSC complete medium, counted using Cellcounter, and 116 and 112 ten thousand cells were harvested in 2T 75 flasks, respectively, without significant difference from fresh microtissue cultured.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A method of isolating mesenchymal stem cells from a placenta, the method comprising the steps of: adding physiological solution containing collagenase I into placenta tissue for digestion for 1-3 h; solid-liquid separation, collecting tissue blocks which are not completely digested; and inoculating the tissue blocks which are not completely digested into a culture medium for wall-attaching culture to obtain mesenchymal stem cells.
2. The method according to claim 1, wherein the collagen I enzyme is present in the physiological solution in an amount of 0.1 to 5% by mass; preferably, the mass percentage is 0.1-1%.
3. The method of claim 1, wherein the physiological solution further comprises 0.000001 to 0.00001% calcium chloride.
4. The method of claim 1, wherein the undigested complete tissue mass is of a size of (200-800) × (200-800) μιη.
5. The method of claim 1, wherein the medium comprises MSC complete medium; and/or the culture medium also contains 5-12% of fetal bovine serum.
6. The method according to claim 1, wherein the mass-to-volume ratio of incompletely digested tissue mass to culture medium is (0.05-2): (10-14) g/mL.
7. A mesenchymal stem cell prepared by the method of any one of claims 1-6;
preferably, the positive rate of at least one of the markers CD90, CD105 and CD73 in the mesenchymal stem cells is greater than 98%.
Preferably, the positive rate of at least one of markers CD34, CD11b, CD19, CD45 and HLA-DR in the mesenchymal stem cells is less than 1%.
8. A preservation method of tissue derived from mesenchymal stem cells, comprising the steps of: after adding frozen stock solution to the tissue block which is not completely digested and prepared by the method of any one of claims 1 to 6, carrying out program cooling and storing the tissue block in liquid nitrogen.
9. The preservation method of claim 8 wherein the programmed cooling comprises the steps of: the temperature is raised to-12 to-15 ℃ after the first gradient cooling to-48 to-52 ℃, and the temperature is lowered to-88 to-92 ℃ after the second gradient cooling; preferably, the first gradient cooling comprises the following steps: after mixing the tissue blocks which are not completely digested with the frozen stock solution, regulating the temperature to be 2-6 ℃, and cooling to 5-7 ℃ at the speed of 0.5-2 ℃/min; then cooling to-48 to-52 ℃ at a speed of 22-28 ℃/min; preferably, the temperature rise is that after the first gradient cooling to-48 to-52 ℃, the temperature is quickly raised to-12 to-15 ℃ at 22-28 ℃/min; preferably, the second gradient cooling means that after the temperature is raised to-12 to-15 ℃, the temperature is reduced to-40 to-50 ℃ at 0.5 to 3 ℃ per minute, and then the temperature is reduced to-80 to-100 ℃ at 8 to 12 ℃ per minute.
10. The method according to claim 8, wherein the frozen stock solution is serum containing 7 to 12% dimethyl sulfoxide.
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