CN110257327A - A kind of isolated culture method of umbilical cord mesenchymal stem cells - Google Patents
A kind of isolated culture method of umbilical cord mesenchymal stem cells Download PDFInfo
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- 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
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Abstract
The present invention relates to a kind of isolated culture methods of umbilical cord mesenchymal stem cells, characterized in that includes the following steps: to acquire umbilical cord sample；Clean umbilical cord sample；Umbilical cord sample slice；The processing of umbilical cord tissue thin slice；Umbilical cord tissue thin slice culture；Digestion process；Cell is collected by centrifugation；Inoculated and cultured is resuspended；Had digestive transfer culture culture；Cellular identification.The present invention can efficient separating funicle mesenchyme stem cell, the probability that tissue grows cell is high, can satisfy clinical application and produces the needs of stem cell preparations in enormous quantities；It is small, high-efficient to be separately cultured workload.
Description
Technical field
It is separately cultured the present invention relates to stem cell more particularly to a kind of method for isolation and culture of umbilical mesenchymal stem cells.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is a kind of adult stem cell, it is present in navel
In some tissues such as band, marrow, fat and Cord blood, there is very strong self-renewing and polyembryony layer differentiation capability.Research shows that
It is significant in efficacy in immunological regulation and in terms of promoting tissue repair, has been largely used to rheumatoid arthritis, lupus erythematosus, shifting
In the clinical research and treatment of a variety of major diseases such as graft versus host disease, liver fibrosis.
Umbilical cord mesenchymal stem cells are extracted in neonatal umbilical cord after breaking navel from term birth, have abundance, no
It injures that body, proliferative capacity are strong, it is low etc. to have the advantages that polyembryony layer differentiation capability, immunogenicity, has been obtained more and more special
The approval and application of industry personage.
At present both at home and abroad common separation method substantially there are two types of:
First is that enzymatic isolation method；Enzymatic isolation method is at high cost, has damage to cell, and obtained primary cell quantity is few, it is desirable to amplification to clinic
The cell quantity of application must carry out the culture of more algebra in vitro, increase the probability of cell mutation, increase clinic and answer
Risk.
Second is that tissue block adherent cultivation；Conventional tissue block adherent cultivation is to shred umbilical cord tissue, then will be thin
Small tissue block one one 's is taped against culture dish bottom, and each culture dish needs to spread several hundred pieces of tissues, although can be to avoid enzyme
Some drawbacks of solution, but heavy workload, the probability that tissue grows cell are low.Separating a small amount of cell can be with for scientific research,
But if reaching the cell number of clinical application or producing stem cell preparations in enormous quantities, conventional tissue block adherent cultivation
Just be not suitable for.
CN 103589683A discloses " a kind of separation method and cultural method of umbilical cord mesenchymal stem cells ".The separation
Method includes: sufficiently to be cleaned the neonatal umbilical cord tissue of health with the PBS buffer solution containing penicillin and streptomysin, removes blood
It is dirty；By umbilical cord scissors at the uniform segment of length, carry out Mechanical Method separation, blunt separation China Tong Shi glue, at the same remove arteria umbilicalis and
Umbilical vein；The magnificent Tong Shi glue of removing is uniformly shredded；The magnificent Tong Shi glue shredded is resuspended with MSCs culture medium, is inoculated in gelatin paving quilt
Culture dish in, be placed in CO2Incubator culture is then centrifuged for separation and obtains tissue block and cell re-suspension liquid.Above-mentioned cultural method
Include: that coating processing is carried out to culture dish, discards gelatin, washed with PBS buffer solution；By the tissue block of separation acquisition and carefully
Born of the same parents' re-suspension liquid is inoculated in culture dish；Had digestive transfer culture is carried out when cell growth is fused to 80%-90%.Unquestionably, this is institute
Belong to a kind of beneficial exploration of technical field.
Summary of the invention
The purpose of the present invention is to provide a kind of method for isolation and culture of umbilical mesenchymal stem cells, can efficiently separate
Umbilical cord mesenchymal stem cells, the probability that tissue grows cell is high, can satisfy clinical application and produces stem cell preparations in enormous quantities
Needs；It is small, high-efficient to be separately cultured workload.
The isolated culture method of a kind of umbilical cord mesenchymal stem cells of the present invention, characterized in that include the following steps:
Step 1 acquires umbilical cord sample；10cm(centimetres of umbilical cord after taking the disconnected navel of term birth) it is put into collecting bottle, navel is added
Band organization protection's liquid, send under conditions of 4-8 DEG C to laboratory, the umbilical cord sample of acquisition must be separated within 48 hours
Operation；
Step 2 cleans umbilical cord sample；Umbilical cord sample is first taken out from collecting bottle, is immersed in 75% alcohol and is sterilized, disinfecting time
It is 20-40 seconds；Then, it with normal saline flushing umbilical cord sample, rinses 2-3 times, removal alcohol is remaining；
Step 3, umbilical cord sample slice；Each 1cm in umbilical cord sample both ends is first cut off, the umbilical cord sample of intermediate 5cm length, edge are taken
The transverse direction of umbilical cord sample is cut into 1mm(millimeters) the umbilical cord tissue thin slice of thickness, then by every umbilical cord tissue thin slice (along umbilical cord sample
Longitudinal direction) be half-and-half cut into two panels；
Step 4, the processing of umbilical cord tissue thin slice；First the umbilical cord tissue thin slice for being half-and-half cut into two panels is placed in physiological saline, is cleaned
Bloodstain on clean umbilical cord tissue thin slice；
Step 5, umbilical cord tissue thin slice culture；Umbilical cord tissue thin slice after cleaning up equably is attached on Tissue Culture Dish,
And sheet glass is covered in the upper surface of umbilical cord tissue thin slice；Then, be added complete culture solution, 37 DEG C, 5%CO2 concentration, saturation it is wet
It is cultivated in the incubator of degree；Every the complete culture solution of replacement in 4 days, and observe culture dish bottom and sheet glass Shang Qi taeniae telarum thin slice
The case where cell growth of surrounding；
Step 6, digestion process；After covering with a large amount of cells on culture dish bottom and sheet glass around umbilical cord tissue thin slice, go
Except umbilical cord tissue thin slice, the cell on culture dish and sheet glass is digested with trypsin solution；Digestion 1-3 minutes；
Cell is collected by centrifugation in step 7；2ml fetal calf serum is added and terminates digestion, collects digestive juice in 50ml centrifuge tube, centrifugation
Collect cell；
Inoculated and cultured is resuspended in step 8；The cell being collected by centrifugation is resuspended with the complete culture solution and is inoculated in culture bottle
Culture；Cultivating obtained cell is P1 for cell；
Step 9, had digestive transfer culture culture；To carry out digestion with trypsase and collect simultaneously after P1 cell length to 80% -95% fusion
Secondary culture, the cell that had digestive transfer culture culture obtains are P2 for cell；The mode of its secondary culture is the ratio that 5 bottles are passed with one bottle
It is passed on；
Step 10, cellular identification；After P2 is for cell length to 80% -95% fusion, digestion is collected cell and is identified.
Further, the ingredient of umbilical cord tissue described in step 1 protection liquid be DMEM/F12 culture medium, D-Hanks liquid,
The volume ratio of streptomysin, penicillin, DMEM/F12 culture medium and D-Hanks liquid is 1:1, penicillin concn 100U/ml, strepto-
Plain concentration is 100g/ml.
Further, physiological saline described in step 4 is the injection physiological saline that mass concentration is 0.9%.
Further, complete culture solution described in step 5 is the DMEM/ containing 5% -15% volumetric concentration fetal calf serum
F12 culture medium.
Further, the mass concentration of trypsin solution described in step 6 is 0.125% -0.5%.
Further, cell, centrifugal force 300-700G are collected by centrifugation described in step 7.
Beneficial effects of the present invention
(1) due to using Jie for allowing two cross sections up and down of every umbilical cord thin sectioned tissue that can touch suitable cell growth
Matter --- down cross-sectional can allow sheet of tissue simultaneously on two media close to culture dish bottom, upper cross section close to sheet glass
Grow cell.The probability that all tissues grow cell can reach 99% or more, to substantially increase the utilization rate of umbilical cord tissue
And separative efficiency.
(2) workload of lock out operation is greatly reduced, the separation used time of every part of umbilical cord is 5-10 minutes, improves work
Make efficiency；
In conclusion the present invention is simpler than the operation of the separation method of other existing umbilical cord mesenchymal stem cells, greatly reduce
The workload of operation can efficiently handle a large amount of umbilical cord sample；The probability that umbilical cord tissue can grow cell has obtained greatly
Raising, 99% tissue can grow cell, can obtain a large amount of umbilical cord mesenchymal stem cells, and it is dry to be more suitable for band mesenchyma
The industrialization and clinical application of cell.
Detailed description of the invention
Fig. 1 is laterally to be cut into 1mm(millimeters along umbilical cord sample) the umbilical cord tissue thin slice schematic diagram of thickness；
Fig. 2 is the schematic diagram that umbilical cord tissue thin slice is cut into two halves；
Fig. 3 is that umbilical cord tissue thin slice is uniformly attached to showing on Tissue Culture Dish and in umbilical cord tissue thin slice upper cover upper glass plate
It is intended to；
Fig. 4 is the state diagram (photo) that a large amount of cells are grown around umbilical cord tissue thin slice；
Fig. 5 is the state diagram (amplification factor: 40X) that P1 covers with culture dish for cell；
Fig. 6 is the cell growth curve figure for taking P2 to do for cell；
Fig. 7 is that (low expression CD34, CD45, HLA-DR, expression rate is lower than 2% for the flow cytometer detection results of umbilical cord mesenchymal stem cells
Standard) curve graph；
Fig. 8 be umbilical cord mesenchymal stem cells flow cytometer detection result (height expression CD90, CD73, CD105, expression rate be higher than 95%
Standard) curve graph；
Fig. 9 is umbilical cord mesenchymal stem cells nude mice by subcutaneous into chondrocyte induction differentiated tissue slice map (alcian blue dyeing, arrow meaning
For cartilaginous tissue)；
Figure 10 is the external adipogenic induction differentiation figure of umbilical cord mesenchymal stem cells (oil red dyeing, arrow meaning are fat drips).
Specific embodiment:
Below with reference to picture, the present invention will be further described.
The isolated culture method of a kind of umbilical cord mesenchymal stem cells of the present invention, characterized in that include the following steps:
Step 1 acquires umbilical cord sample；10cm(centimetres of umbilical cord after taking the disconnected navel of term birth) it is put into collecting bottle, navel is added
Band organization protection's liquid, send under conditions of 4-8 DEG C to laboratory, the umbilical cord sample of acquisition must be separated within 48 hours
Operation；
The ingredient of the umbilical cord tissue protection liquid is DMEM/F12 culture medium, D-Hanks liquid, streptomysin, penicillin, DMEM/
The volume ratio of F12 culture medium and D-Hanks liquid is 1:1, and penicillin concn 100U/ml, streptomysin concentration is 100g/ml.
Step 2 cleans umbilical cord sample；Umbilical cord sample is first taken out from collecting bottle, is immersed in 75% alcohol and sterilizes, and is sterilized
Time is 20-40 seconds；Then, it with normal saline flushing umbilical cord sample, rinses 2-3 times, removal alcohol is remaining；
Step 3, umbilical cord sample slice；Each 1cm in umbilical cord sample both ends is first cut off, the umbilical cord sample of intermediate 5cm length, edge are taken
The transverse direction of umbilical cord sample is cut into 1mm(millimeters) the umbilical cord tissue thin slice (referring to Fig. 1) of thickness, then by every umbilical cord tissue thin slice
(along the longitudinal direction of umbilical cord sample) is half-and-half cut into two panels (referring to fig. 2)；
Step 4, the processing of umbilical cord tissue thin slice；First the umbilical cord tissue thin slice for being half-and-half cut into two panels is placed in physiological saline, is cleaned
Bloodstain on clean umbilical cord tissue thin slice；
The physiological saline is the injection physiological saline that mass concentration is 0.9%.
Step 5, umbilical cord tissue thin slice culture；Umbilical cord tissue thin slice after cleaning up equably is attached to cell culture
On ware, and cover sheet glass in the upper surface of umbilical cord tissue thin slice (referring to Fig. 3)；Then, complete culture solution is added, in 37 DEG C, 5%
CO2 concentration, saturated humidity incubator in cultivate；Every the complete culture solution of replacement in 4 days, and observe culture dish bottom and sheet glass
The case where cell around Shang Qi taeniae telarum thin slice is grown；One piece of umbilical cord tissue piece can be allowed in upper and lower two kinds of suitable cells in this way
Cell is grown simultaneously in the medium of growth, efficiency is greatly improved.
The complete culture solution is the DMEM/F12 culture medium containing 5% -15% volumetric concentration fetal calf serum.
Step 6, digestion process；A large amount of cells are covered on culture dish bottom and sheet glass around umbilical cord tissue thin slice
Afterwards (referring to fig. 4), umbilical cord tissue thin slice is removed, the cell on culture dish and sheet glass is digested with trypsin solution；
Digest 1-3 minutes (specific digestion time is within this range depending on microscopically observation situation)；The trypsase is molten
The mass concentration of liquid is 0.125% -0.5%；
Cell is collected by centrifugation in step 7；2ml fetal calf serum is added and terminates digestion, collects digestive juice in 50ml centrifuge tube, centrifugation
Collect cell；Described is collected by centrifugation cell, centrifugal force 300-700G；
Inoculated and cultured is resuspended in step 8；The cell being collected by centrifugation is resuspended with the complete culture solution and is inoculated in culture bottle
Culture；Cultivating obtained cell is P1 for cell；
Step 9, had digestive transfer culture culture；With (referring to Fig. 5) after P1 cell length to 80% -95% fusion, carried out with trypsase
Digestion is collected and secondary culture, and the cell that had digestive transfer culture culture obtains is P2 for cell；The mode of its secondary culture is with one bottle
Five bottles of ratio is passed to be passed on；
Step 10, cellular identification；After P2 is for cell length to 80% -95% fusion, digestion is collected cell and is identified.
It takes a part of cell to be cultivated and makes cell growth curve, as a result referring to Fig. 6；
It takes a part of cell to carry out flow cytometry identification umbilical cord mesenchymal stem cells surface marker expression characteristic, has detected altogether
Six cell surface marker objects, are CD34, CD45, HLA-DR, CD73, CD105, CD90 respectively, testing result is low expression
CD34, CD45, HLA-DR, expression value is 2% hereinafter, referring to Fig. 7；Height expression CD73, CD105, CD90, expression value 95% with
On, referring to Fig. 8；Meet the standard of mescenchymal stem cell；
A part of cell is taken to do Osteoinductive differentiation in external adipogenic induction Analytical Chemical Experiment (result is referring to Figure 10) and nude mouse real
Test (result is referring to Fig. 9).
Claims (6)
1. a kind of isolated culture method of umbilical cord mesenchymal stem cells, characterized in that include the following steps:
Step 1 acquires umbilical cord sample；10cm(centimetres of umbilical cord after taking the disconnected navel of term birth) it is put into collecting bottle, navel is added
Band organization protection's liquid, send under conditions of 4-8 DEG C to laboratory, the umbilical cord sample of acquisition must be separated within 48 hours
Operation；
Step 2 cleans umbilical cord sample；Umbilical cord sample is first taken out from collecting bottle, is immersed in 75% alcohol and is sterilized, disinfecting time
It is 20-40 seconds；Then, it with normal saline flushing umbilical cord sample, rinses 2-3 times, removal alcohol is remaining；
Step 3, umbilical cord sample slice；Each 1cm in umbilical cord sample both ends is first cut off, the umbilical cord sample of intermediate 5cm length, edge are taken
The transverse direction of umbilical cord sample is cut into 1mm(millimeters) the umbilical cord tissue thin slice of thickness, then every umbilical cord tissue thin slice is half-and-half cut into two
Piece；
Step 4, the processing of umbilical cord tissue thin slice；First the umbilical cord tissue thin slice for being half-and-half cut into two panels is placed in physiological saline, is cleaned
Bloodstain on clean umbilical cord tissue thin slice；
Step 5, umbilical cord tissue thin slice culture；Umbilical cord tissue thin slice after cleaning up equably is attached on Tissue Culture Dish,
And sheet glass is covered in the upper surface of umbilical cord tissue thin slice；Then, be added complete culture solution, 37 DEG C, 5%CO2 concentration, saturation it is wet
It is cultivated in the incubator of degree；Every the complete culture solution of replacement in 4 days, and observe culture dish bottom and sheet glass Shang Qi taeniae telarum thin slice
The case where cell growth of surrounding；
Step 6, digestion process；After covering with a large amount of cells on culture dish bottom and sheet glass around umbilical cord tissue thin slice, go
Except umbilical cord tissue thin slice, the cell on culture dish and sheet glass is digested with trypsin solution；Digestion 1-3 minutes；
Cell is collected by centrifugation in step 7；2ml fetal calf serum is added and terminates digestion, collects digestive juice in 50ml centrifuge tube, centrifugation
Collect cell；
Inoculated and cultured is resuspended in step 8；The cell being collected by centrifugation is resuspended with the complete culture solution and is inoculated in culture bottle
Culture；Cultivating obtained cell is P1 for cell；
Step 9, had digestive transfer culture culture；To carry out digestion with trypsase and collect simultaneously after P1 cell length to 80% -95% fusion
Secondary culture, the cell that had digestive transfer culture culture obtains are P2 for cell；The mode of its secondary culture is the ratio that 5 bottles are passed with one bottle
It is passed on；
Step 10, cellular identification；After P2 is for cell length to 80% -95% fusion, digestion is collected cell and is identified.
2. the isolated culture method of umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that: described in step 1
Umbilical cord tissue protection liquid ingredient be DMEM/F12 culture medium, D-Hanks liquid, streptomysin, penicillin, DMEM/F12 culture medium
Volume ratio with D-Hanks liquid is 1:1, and penicillin concn 100U/ml, streptomysin concentration is 100g/ml.
3. the isolated culture method of umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that: described in step 4
Physiological saline be mass concentration be 0.9% injection physiological saline.
4. the isolated culture method of umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that: described in step 5
Complete culture solution be the DMEM/F12 culture medium containing 5% -15% volumetric concentration fetal calf serum.
5. the isolated culture method of umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that: described in step 6
Trypsin solution mass concentration be 0.125% -0.5%.
6. the isolated culture method of umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that: described in step 7
Cell, centrifugal force 300-700G is collected by centrifugation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
| CN113528344A (en) * | 2021-07-22 | 2021-10-22 | 青岛思拓新源细胞医学有限公司 | Umbilical cord tissue mesenchymal stem cell culture assembly and use method thereof |
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| CN113652397A (en) * | 2020-05-12 | 2021-11-16 | 辽宁医学诊疗科技研发中心有限公司 | Culture method of animal-origin-free umbilical cord mesenchymal stem cells |
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