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CN108795855A - A kind of serum free medium of mescenchymal stem cell - Google Patents

  • ️Tue Nov 13 2018

CN108795855A - A kind of serum free medium of mescenchymal stem cell - Google Patents

A kind of serum free medium of mescenchymal stem cell Download PDF

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Publication number
CN108795855A
CN108795855A CN201810671308.7A CN201810671308A CN108795855A CN 108795855 A CN108795855 A CN 108795855A CN 201810671308 A CN201810671308 A CN 201810671308A CN 108795855 A CN108795855 A CN 108795855A Authority
CN
China
Prior art keywords
stem cell
concentration
cell
mescenchymal stem
growth factor
Prior art date
2018-06-26
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810671308.7A
Other languages
Chinese (zh)
Inventor
李首
李首一
石晓川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jilin Sun Bird Regeneration Medical Engineering Co Ltd
Original Assignee
Jilin Sun Bird Regeneration Medical Engineering Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
2018-06-26
Filing date
2018-06-26
Publication date
2018-11-13
2018-06-26 Application filed by Jilin Sun Bird Regeneration Medical Engineering Co Ltd filed Critical Jilin Sun Bird Regeneration Medical Engineering Co Ltd
2018-06-26 Priority to CN201810671308.7A priority Critical patent/CN108795855A/en
2018-11-13 Publication of CN108795855A publication Critical patent/CN108795855A/en
Status Pending legal-status Critical Current

Links

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Abstract

The present invention relates to stem cell fields, disclose a kind of culture medium and its large-scale cultivation method of mescenchymal stem cell.The culture medium of mescenchymal stem cell of the present invention, including serum free medium, basic fibroblast growth factor, epithelical cell growth factor, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, astragalus polyose, vitamin, insulin, hydrocortisone, fibronectin and linoleic acid.Culture medium of the present invention does not contain FBS, reduces the probability of cell contamination, while playing facilitation to the amplification of MSC.Large-scale culture is carried out to mescenchymal stem cell using culture medium of the present invention and can get a large amount of high-purity mescenchymal stem cells, and the good stem cell properties of mescenchymal stem cell can be kept, is suitable for the large-scale culture of all types MSC.

Description

A kind of serum free medium of mescenchymal stem cell

Technical field

The present invention relates to stem cell fields, and in particular to a kind of serum free medium of mescenchymal stem cell.

Background technology

Mescenchymal stem cell (MSC, mesenchymal stem cells) is the important member of stem cell line, is derived from The mesoderm of mesoderm growing early stage belongs to multipotential stem cell, and MSC initially has found in marrow, because it is with multi-lineage potential, hematopoiesis Support and promote stem cell implantation, immunoregulation and the concern that people are increasingly subject to the features such as self-replacation.As mesenchyma is dry Cell is in vivo or in vitro under specific inductive condition, can be divided into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, The Various Tissues cell such as cardiac muscle, endothelium still has multi-lineage potential, can be used as ideal after continuous passage culture and freezen protective Seed cell for injuries of tissues and organs reparation caused by aging and lesion.Mescenchymal stem cell clinical application at present is in solution A variety of diseases in the blood system, angiocardiopathy, hepatic sclerosis, the nervous system disease, meniscus of knee joint part cut off injury repair, Autoimmune disease etc. achieves important breakthrough, has saved the life of more sufferers.In addition, mescenchymal stem cell is in god There is long-range development prospect through system reparation and more aspects.

Numerous studies confirm that the mescenchymal stem cell in adult can also be continuously decreased with the increase at age, content, and And proliferation and the ability of differentiation can also decline accordingly.Therefore it needs to advise isolated primary mescenchymal stem cell greatly Mould culture efficient amplification, with the technical problem for overcoming mescenchymal stem cell cell quantity few.

Currently, in the routine culture of mescenchymal stem cell, most common cultivating system is to use DMEM/F12 culture mediums In addition the fetal calf serum (FBS) that volume ratio is 10% carries out in vitro culture and the amplification of mescenchymal stem cell.Between although FBS can be Mesenchymal stem cells provide necessary nutriment, but FBS belongs to animal derived substance, and comparison of ingredients is complicated, in clinical application There are potential risks, also increase the probability of cell contamination.And FBS is a kind of biological products that price is relatively high, In the large-scale culture of mesenchymal stem cells, the cost of production can be increased.

Invention content

In view of this, present invention aims in view of the problems of the existing technology, a kind of mescenchymal stem cell is provided Culture medium does not contain FBS, reduces the probability of cell contamination, while playing facilitation to the amplification of MSC.In order to realize this hair Bright purpose, the present invention adopt the following technical scheme that：

A kind of culture medium of mescenchymal stem cell, including serum free medium, basic fibroblast growth factor, epidermis Porcine HGF, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, astragalus polyose, Vitamin, insulin, hydrocortisone, fibronectin and linoleic acid.

It is thin that the culture medium of mescenchymal stem cell of the present invention adds basic fibroblast on the basis of serum free medium The intracellular growth factor, epithelical cell growth factor, platelet derived growth factor PDGF, transforming growth factor-β, transferrins, L- paddy ammonia Phthalein amine, astragalus polyose, vitamin, insulin, hydrocortisone, fibronectin and linoleic acid do not contain FBS, and it is dirty to reduce cell The probability of dye, while facilitation is played to the amplification of MSC, while keeping original mescenchymal stem cell characteristic.

Wherein, preferably, serum free medium described in the culture medium of the mescenchymal stem cell is lonza UltraCULTURE MEDIUM serum free mediums.

Preferably, a concentration of 1- of basic fibroblast growth factor described in the culture medium of the mescenchymal stem cell 100ng/m L, a concentration of 5-20ng/m L of epithelical cell growth factor, a concentration of 5- of platelet derived growth factor PDGF 10 μ g/L, a concentration of 1-100ng/m L of transforming growth factor-β, the transferrin concentrations are 5-20mg/L, the L- Paddy ammonia phthalein amine concentration is 1-5mM, and a concentration of 40-80mg/L of astragalus polyose, the vitamine concentration is 50-100mg/L, institute It is 1-10U/ml, a concentration of 5-20mg/L of hydrocortisone, a concentration of 5-50mg/ of fibronectin to state insulin concentration L, linoleic acid 2-10mg/L.

In some preferred embodiments, a concentration of 50ng/m L of the basic fibroblast growth factor.

In some preferred embodiments, a concentration of 5ng/mL of the epithelical cell growth factor.

In some preferred embodiments, a concentration of 5ng/mL of the platelet derived growth factor.

In some preferred embodiments, a concentration of 20ng/m L of the transforming growth factor-β.

In some preferred embodiments, the transferrin concentrations are 10mg/L.

In some preferred embodiments, the L- paddy ammonia phthalein amine concentration is 2mM.

In some preferred embodiments, a concentration of 50mg/L of the astragalus polyose.

In some preferred embodiments, the vitamin is water soluble vitamin, more preferably vitamin C.The dimension Raw element concentration is preferably 60mg/L.

In some preferred embodiments, the insulin concentration is 5U/ml.

In some preferred embodiments, a concentration of 10mg/L of the hydrocortisone.

In some preferred embodiments, a concentration of 20mg/L of the fibronectin.

In some preferred embodiments, the linoleic acid 5mg/L.

The present invention also provides a kind of large-scale cultivation methods of mescenchymal stem cell, and P1 is taken to be inoculated with for mescenchymal stem cell The culture medium of above-mentioned mescenchymal stem cell, is placed in 5%CO2, 37 DEG C of incubator cultures, when cell fusion degree reaches 80%~90% After pass on.

The large-scale cultivation method of mescenchymal stem cell of the present invention is suitable for the large-scale culture of all types MSC, The mescenchymal stem cell can be mesenchymal stem cell, fat mesenchymal stem cell, umbilical cord mesenchymal stem cells, placenta Mescenchymal stem cell, dental pulp mescenchymal stem cell or peripheral blood mescenchymal stem cell.

Preferably, the inoculum density is 1 × 105/mL-2×106/ mL P1 are for mescenchymal stem cell.More preferably 5 ×105/mL。

In some embodiments, the P1 is specially that tissue block is cut for the primary separation and culture method of mescenchymal stem cell It is broken, complete medium, in 37 DEG C, 5%CO is added2During which static gas wave refrigerator adds complete medium, wait for that tissue block has cell to climb out of After remove tissue block, complete medium is added after cleaning and continues to cultivate, after cell fusion degree reaches 80%~90%, removes thin Born of the same parents' culture supernatant is added after cleaning and is digested containing tryptic digestive juice, and complete medium terminates digestion, and centrifugation is abandoned supernatant, is finished Full culture medium is resuspended.

In some embodiments, tryptose is contained described in primary separation and culture methods of the P1 for mescenchymal stem cell Trypsinase concentration is 0.25v/v%, contained a concentration of 0.04v/v% of EDTA in enzymic digestion liquid.

In some embodiments, primary separation described in primary separation and culture methods of the P1 for mescenchymal stem cell The time that digestive juice digests in cultural method is 1-2min.

In some embodiments, mesenchyma described in primary separation and culture methods of the P1 for mescenchymal stem cell is dry Cell origin and umbilical cord tissue.The umbilical cord can be derived from the umbilical cord tissue thrown aside in postpartum, be preserved using dual anti-PBS is contained. It is described containing dual anti-PBS be the phosphate buffer containing penicillin and streptomysin, wherein penicillin working concentration be 100U/ ML, streptomysin working concentration are 0.1mg/mL.

In some embodiments, the addition of complete medium is 0.07mL/cm before the culture2-0.1mL/cm2。

In some preferred embodiments, it needs to add complete medium in the training period until tissue block has cell to climb Go out.In certain embodiments, the addition that complete medium is added during the culture is 0.07mL/cm2-0.1mL/ cm2。

P1 of the present invention is gone in the primary separation and culture method of mescenchymal stem cell after tissue block has cell to climb out of Fall tissue block, and cell is cleaned.PBS cleanings may be used in the cleaning.

Complete medium is added in the primary separation and culture method of mescenchymal stem cell in P1 of the present invention after cleaning Continue to cultivate, until cell fusion degree reaches 80%~90%.In some embodiments, after the cleaning complete medium plus It is 0.15mL/cm to enter amount2-0.21mL/cm2。

In some embodiments, P1 of the present invention is described in the primary separation and culture method of mescenchymal stem cell The addition for terminating the complete medium of digestion is 5 times~10 times of digestive juice volume.

In some embodiments, described in primary separation and culture methods of the P1 of the present invention for mescenchymal stem cell from The heart is that 200g-400g centrifuges 5min.

In a specific embodiment, between the large-scale cultivation method culture for investigating mescenchymal stem cell of the present invention The proliferative capacity of mesenchymal stem cells, the large-scale culture side of mescenchymal stem cell of the present invention known to cell growth curve figure The mescenchymal stem cell 0~2 day of method culture is the laundering period, and 3~6 days are exponential phase, and cell Proliferation is very fast, the 7th day cell It is proliferated slack-off, enters plateau.Show the mesenchyma of the large-scale cultivation method culture of mescenchymal stem cell of the present invention The proliferative capacity of stem cell is stronger.

Further, in a specific embodiment, the present invention uses Western blot mesenchyma of the present invention Surface marker CD34, CD45, CD44, CD90's of the mescenchymal stem cell cell of the large-scale cultivation method culture of stem cell As a result expression shows the mescenchymal stem cell P10 of the large-scale cultivation method culture of mescenchymal stem cell of the present invention The expression rate of the cell surface marker in generation has no apparent change compared with P1 is for the expression rate of cell, is higher level Expression.Show that the mescenchymal stem cell of the large-scale cultivation method culture of mescenchymal stem cell of the present invention can be kept very well The form of stem cell and good stem cell properties.

The culture medium of mescenchymal stem cell of the present invention, including serum free medium, basic fibroblast growth because Son, epithelical cell growth factor, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, Huang Astragalus polysaccharides, vitamin, insulin, hydrocortisone, fibronectin and linoleic acid.Culture medium of the present invention does not contain FBS, drop The probability of low cell contamination, while facilitation is played to the amplification of MSC.It is dry to mesenchyma thin using culture medium of the present invention Born of the same parents carry out large-scale culture and can get a large amount of high-purity mescenchymal stem cells, and can keep the good stem cell of mescenchymal stem cell Characteristic is suitable for the large-scale culture of all types MSC.

Description of the drawings

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technology description to be briefly described.

Fig. 1 shows the cytological map of P10 after 6 secondary culture of embodiment for umbilical cord MSC, wherein figure a is cells of the P1 for umbilical cord MSC Figure, figure b are cytological maps of the P10 for umbilical cord MSC, and amplification factor is 100 times；

Fig. 2 shows that P10 is for the growth curve chart of umbilical cord MSC after 6 secondary culture of embodiment.

Specific implementation mode

Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described, Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all Belong to the scope of protection of the invention.

Embodiment 1, mescenchymal stem cell culture medium of the present invention

A kind of culture medium of mescenchymal stem cell, including serum free medium, basic fibroblast growth factor, epidermis Porcine HGF, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, astragalus polyose, Vitamin C, insulin, hydrocortisone, fibronectin and linoleic acid.The wherein described serum free medium is lonza UltraCULTURE MEDIUM serum free mediums, a concentration of 100ng/m L of basic fibroblast growth factor, institute State epithelical cell growth factor a concentration of 5ng/m L, a concentration of 10 μ g/L of platelet derived growth factor PDGF, the conversion life Long a concentration of 10ng/m L of the factor-β, the transferrin concentrations are 20mg/L, and the L- paddy ammonia phthalein amine concentration is 1mM, described A concentration of 80mg/L of astragalus polyose, the vitamin C concentration are 50mg/L, and the insulin concentration is 10U/ml, the hydrogenation Cortisone a concentration of 5mg/L, a concentration of 50mg/L of the fibronectin, linoleic acid 2mg/L.

Embodiment 2, mescenchymal stem cell culture medium of the present invention

A kind of culture medium of mescenchymal stem cell, including serum free medium, basic fibroblast growth factor, epidermis Porcine HGF, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, astragalus polyose, Vitamin C, insulin, hydrocortisone, fibronectin and linoleic acid.The wherein described serum free medium is lonza UltraCULTURE MEDIUM serum free mediums, a concentration of 10ng/m L of basic fibroblast growth factor are described Epithelical cell growth factor a concentration of 20ng/mL, a concentration of 5 μ g/L of platelet derived growth factor PDGF, the conversion growth A concentration of 100ng/m L of the factor-β, the transferrin concentrations are 5mg/L, and the L- paddy ammonia phthalein amine concentration is 5mM, the Huang A concentration of 40mg/L of astragalus polysaccharides, the vitamin C concentration are 100mg/L, and the insulin concentration is 1U/ml, and the hydrogenation can Pine a concentration of 20mg/L, a concentration of 5mg/L of the fibronectin, linoleic acid 10mg/L.

Embodiment 3, mescenchymal stem cell culture medium of the present invention

A kind of culture medium of mescenchymal stem cell, including serum free medium, basic fibroblast growth factor, epidermis Porcine HGF, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, astragalus polyose, Vitamin C, insulin, hydrocortisone, fibronectin and linoleic acid.The wherein described serum free medium is lonza UltraCULTURE MEDIUM serum free mediums, a concentration of 50ng/m L of basic fibroblast growth factor are described Epithelical cell growth factor a concentration of 5ng/m L, a concentration of 5 μ g/L of platelet derived growth factor PDGF, the conversion growth A concentration of 20ng/m L of the factor-β, the transferrin concentrations are 10mg/L, and the L- paddy ammonia phthalein amine concentration is 2mM, the Huang A concentration of 50mg/L of astragalus polysaccharides, the vitamin C concentration be 60mg/L, the insulin concentration be 5U/ml, the hydrogenation can Pine a concentration of 10mg/L, a concentration of 20mg/L of the fibronectin, linoleic acid 5mg/L.

Primary separation and the culture of embodiment 4, umbilical cord mesenchymal stem cells

Ox umbilical cord tissue block is obtained, shreds, complete medium is added in tissue block, cell training is transferred to after piping and druming uniformly It supports in ware；Rocking culture dish makes tissue block be uniformly distributed in culture dish, is transferred to 5%CO2, train in 37 DEG C of cell incubators It supports, and routine observation cell growth status；After cell climbs out of, old culture medium and tissue block are discarded, culture dish is cleaned with PBS Afterwards, addition complete medium, which continues to be placed in cell incubator, cultivates；After cell fusion degree reaches 80%~90%, discard old Culture medium, after cleaning culture dish with PBS, the digestive juice that trypsase containing 0.25v/v% and 0.04v/v%EDTA is added disappears Change 1~2min, after complete medium termination digestion is added, by cell piping and druming at single cell suspension, obtains umbilical cord MSC, and be denoted as P1 is for cell.

The large-scale culture of embodiment 5, umbilical cord MSC

After the P1 obtained in embodiment 4 is collected by centrifugation for cell, cell is carried out using Count Star cell counters It counts, 5 × 10 is adjusted to according to count results culture medium described in embodiment 35The inoculum density of/mL, and it is inoculated into 15cm's In big culture dish, it is placed in 5%CO2, 37 DEG C of incubators continue to cultivate.It can be carried out after cell fusion degree reaches 80%~90% Pass on again, and be denoted as P2 for cell, and so on repeat passage carry out extensive amplification cultivation can when reaching P10 for cell Largely met the MSC of clinical treatment dosage.

Embodiment 6,

From morphological observation, ability of cell proliferation and flow cytometer detection cell surface marker, to the P10 generations after continuous passage Umbilical cord MSC carries out the identification of cell：

(1) MSC morphological observations

Embodiment 5 is observed under inverted microscope per the growing state and metamorphosis of generation umbilical cord MSC, compares P10 for MSC Form and P1 for, whether there is or not changing, the state outcome of preliminary judgement cell is shown in Fig. 1 between MSC.

As seen from Figure 1, umbilical cord MSC P1 are preferable for cell state, are fusiformis, at threadiness, it is dry thin to meet mesenchyma The Morphological Features of born of the same parents.Umbilical cord MSC P10 are also mostly fusiformis from the perspective of morphology into threadiness for cell, the nothing compared with P1 is for cell Apparent variation.

(2) growth curve of MSC proliferative capacities

The P10 obtained in Example 5 is for cell, and the culture medium described in embodiment 3 is by 5 × 10 after being digested using pancreatin5/ ML is inoculated on 24 orifice plates, per hole 1mL systems.It is collected every taking three hole cells to carry out pancreatin digestion and cell respectively for 24 hours, And carry out the average value that cell count calculates three hole cell quantities.It is carried out continuously cell count seven days, further according to experimental result, The growth curve for drawing out the cell, is as a result shown in Fig. 2.

The strong MSC cells of general proliferative capacity, growth curve can be in " S " type, can be divided into 3 growth periods：First stage For the laundering period, cell Proliferation is slower：Second stage is exponential phase, and cell proliferation rate becomes faster, and is in logarithmic growth；Third rank Section is plateau, cells proliferation slowed down.According to the cell growth curve figure in umbilical cord MSC seven days generations of P10, it can be seen that be within 0~2 day Laundering period, 3~6 days are exponential phase, and cell Proliferation is very fast, and cell Proliferation is slack-off within the 7th day, enters plateau.It is thin from Fig. 2 Intracellular growth curve graph is it is found that the proliferative capacity of the cell is stronger.

(3) flow cytometer detection MSC surface markers

The umbilical cord MSC cells in P10 generations are collected, single cell suspension is made in digestion after collecting, be added anti-with immunofluorescence Body is incubated, and the flow cytometer detection of umbilical cord MSC surface markers is carried out.The specific antibody of addition is respectively that CD34 (is negative Expression), CD45 (be negative expression), CD44 (positive expression), CD90 (positive expression).

As a result the expression that umbilical cord MSC P1 are 97.9%, CD45-CD90+ for the expression rate of cell CD34-CD44+ is shown Rate is up to 97.4%；And the expression rate that umbilical cord MSC P10 are 98.1%, CD45-CD90+ for the expression rate of cell CD34-CD44+ It is 96.3%.The result shows that the expression rate of the cell surface marker in P10 generations has no apparent compared with P1 is for the expression rate of cell Change, be the expression of higher level.

Culture medium described in embodiment 1 and embodiment 2 is respectively adopted according to the method for embodiment 5, P1 generations are obtained to embodiment 4 Cell is cultivated, and morphological observation, ability of cell proliferation and flow cytometer detection cell surface mark are carried out according to the method for embodiment 6 The identification of will object, it is as a result similar to Example 6, the P10 of culture in mescenchymal stem cell form compared with P1 is for cell without bright Aobvious variation, culture P10 is stronger for the proliferative capacity of mescenchymal stem cell, and can keep the form of stem cell and good very well Stem cell properties.

Claims (9)

1. a kind of culture medium of mescenchymal stem cell, including serum free medium, basic fibroblast growth factor, epidermis are thin The intracellular growth factor, platelet derived growth factor PDGF, Peritoneal fibrosis β, transferrins, L- paddy ammonia phthaleins amine, astragalus polyose, dimension Raw element, insulin, hydrocortisone, fibronectin and linoleic acid.

2. culture medium according to claim 1, which is characterized in that the serum free medium is lonza UltraCULTURE MEDIUM serum free mediums.

3. culture medium according to claim 1, which is characterized in that a concentration of 1- of basic fibroblast growth factor 100ng/m L, a concentration of 5-20ng/m L of epithelical cell growth factor, a concentration of 5- of platelet derived growth factor PDGF 10 μ g/L, a concentration of 1-100ng/m L of transforming growth factor-β, the transferrin concentrations are 5-20mg/L, the L- Paddy ammonia phthalein amine concentration is 1-5mM, and a concentration of 40-80mg/L of astragalus polyose, the vitamine concentration is 50-100mg/L, institute It is 1-10U/ml, a concentration of 5-20mg/L of hydrocortisone, a concentration of 5-50mg/ of fibronectin to state insulin concentration L, the linoleic acid 2-10mg/L.

4. a kind of large-scale cultivation method of mescenchymal stem cell, take P1 between described in mescenchymal stem cell inoculation claim 1 The culture medium of mesenchymal stem cells, is placed in 5%CO2, 37 DEG C of incubator cultures, passed after cell fusion degree reaches 80%~90% Generation.

5. large-scale cultivation method according to claim 4, inoculum density is 1 × 105/mL-2×106/ mL fills between P1 generations Matter stem cell.

6. large-scale cultivation method according to claim 4, the mescenchymal stem cell is mesenchymal stem cell, fat It is filled between fat mescenchymal stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, dental pulp mescenchymal stem cell or peripheral blood Matter stem cell.

7. according to the large-scale cultivation method described in claim 4-6 any one, the P1 is for the primary of mescenchymal stem cell Isolated culture method is specially that tissue block shreds, and complete medium, in 37 DEG C, 5%CO is added2During which static gas wave refrigerator has been added Full culture medium removes tissue block after tissue block has cell to climb out of, and complete medium is added after cleaning and continues to cultivate, when cell melts It is right reach 80%~90% after, remove cells and supernatant, after cleaning be added containing tryptic digestive juice digest, completely cultivate Base terminates digestion, and centrifugation is abandoned supernatant, is resuspended with complete medium.

8. large-scale cultivation method according to claim 7 contains trypsase described in the primary separation and culture method Trypsinase concentration is 0.25v/v%, contained a concentration of 0.04v/v% of EDTA in digestive juice.

9. large-scale cultivation method according to claim 7, in the primary separation and culture method digestive juice digest when Between be 1-2min.

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CN109402051A (en) * 2018-12-28 2019-03-01 青岛麦迪赛斯生物科技有限公司 A kind of human umbilical cord mesenchymal stem cells serum free medium
CN110172440A (en) * 2019-04-18 2019-08-27 青岩生物科技(湖州)有限公司 It is a kind of for cultivating the serum free medium of mesodermal stroma cell
CN110551685A (en) * 2019-10-10 2019-12-10 陕西佰傲干细胞再生医学有限公司 Cell culture solution and application thereof
CN112011506A (en) * 2020-09-16 2020-12-01 郑州佐爵生物科技有限公司 Method for promoting in-vitro proliferation of mesenchymal stem cells
CN113249313A (en) * 2021-05-14 2021-08-13 达瑟儿(上海)生命科技有限公司 Primary culture method of stem cells with high cell adherence capacity
CN115044530A (en) * 2022-05-19 2022-09-13 唐颐控股(深圳)有限公司 Engineered microcarrier and preparation method and application thereof
CN115044530B (en) * 2022-05-19 2024-02-09 唐颐控股(深圳)有限公司 Engineered microcarrier and preparation method and application thereof
CN116042518A (en) * 2023-01-17 2023-05-02 上海利康瑞生物工程有限公司 Novel serum-free medium for mesenchymal stem cells and preparation method thereof
CN117448268A (en) * 2023-12-25 2024-01-26 深圳市北科生物科技有限公司 Serum-free culture medium of dental pulp mesenchymal stem cells and culture method thereof
CN117448268B (en) * 2023-12-25 2024-05-10 深圳市北科生物科技有限公司 Serum-free culture medium of dental pulp mesenchymal stem cells and culture method thereof
CN117802039A (en) * 2024-03-01 2024-04-02 泉美智能科技(山东)有限公司 Serum-free mesenchymal stem cell three-dimensional culture medium and application thereof

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