CN106479970A - A kind of method of large-scale culture human adipose mesenchymal stem cells - Google Patents

A kind of method of large-scale culture human adipose mesenchymal stem cells Download PDF

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CN106479970A
CN106479970A CN201611050980.1A CN201611050980A CN106479970A CN 106479970 A CN106479970 A CN 106479970A CN 201611050980 A CN201611050980 A CN 201611050980A CN 106479970 A CN106479970 A CN 106479970A
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stem cell
mesenchymal stem
microcarrier
fat
culture medium
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葛啸虎
陈海佳
王飞
王一飞
黄幸
王小燕
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers

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Abstract

The present invention relates to stem cell field, discloses a kind of method of large-scale culture human adipose mesenchymal stem cells.The method of large-scale culture human adipose mesenchymal stem cells of the present invention adds fat mesenchymal stem cell culture medium in glass blake bottle, in humidity 95%, 5%CO2Incubator in 35 DEG C incubate balance 30 minutes；The temperature to 35 DEG C of adjustment fat mesenchymal stem cell culture medium, pH7.05, microcarrier and the fat mesenchymal stem cell of pretreatment is added, 5RPM～20RPM stirs 2h；37 DEG C are warming up in 15RPM 40RPM, 5%CO2Under the conditions of cultivate 96h, period adds fat mesenchymal stem cell culture medium.Compared with the conventional method, cell is obtained more, cell viability and multiplication capacity are strong, and can keep the form of fat mesenchymal stem cell and good stem cell properties very well after the method propagation of large-scale culture human adipose mesenchymal stem cells of the present invention.

Description

A kind of method of large-scale culture human adipose mesenchymal stem cells
Technical field
The present invention relates to stem cell field, and in particular to a kind of method of large-scale culture human adipose mesenchymal stem cells.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is derived from the mesoderm of mesoderm growing early stage with outward Germinal layer, the cell with characteristics such as self, Multidirectional Differentiation, pluripotency, hematopoiesis support and immunoregulations, produces Give birth to and nutritional substance is discharged, cell repair and Angiogenesiss are promoted by paracrine approach, is had in regenerative medicine field important Effect.
At present, mescenchymal stem cell is mainly extracted from fat, marrow, umbilical cord, placenta tissue.Mescenchymal stem cell is continuous Still there is multi-lineage potential after Secondary Culture and freezen protective, be the various diseases of clinical treatment, such as the nervous system disease, kidney The treatment of disease, autoimmune disease, malignant tumour etc. provides new approach.
Fat mesenchymal stem cell (ADMSCs) is a kind of stem cell being present in human fat tissue.From fat Mescenchymal stem cell, draws materials conveniently, abundance, it is easy to which collection and transport, biological characteristics are stable, and immunogenicity is low, is as good as Body rejection, low cost, harmless to donor and be not related to the advantages such as ethics problem so as to become following stem cell in doctor Treat the ideal chose with great potential in application.
But fat mesenchymal stem cell is either used for clinical or is required for substantial amounts of cell for organizational project.Face Bed application often needs to reach 108-1010The order of magnitude.As mescenchymal stem cell needs adherent growth, the cell quantity of this scale If expanded using blake bottle in the lab, consumptive material consumption is big, and artificial pays and surprising.
Bioreactor is the biological function having using organism, in vitro or in vivo by biochemical reaction or biology The apparatus system of the metabolism acquisition target product of itself, cell, histoorgan etc..Bioreactor can be used for extensive amplification Cell, but generally bioreactor is used for the extensive amplification of suspension cell, and be open system, need a lot of support Equipment, not only complex operation, is also easy to the problems such as causing pollution, and clinical practice has sizable risk.
Content of the invention
In view of this, present invention aim at the problem existed for prior art, provides a kind of large-scale culture people fat The cultural method of the method fat mesenchymal stem cell of fat mescenchymal stem cell, the method for the invention can be obtained the short time in a large number High-purity fat mesenchymal stem cell, and the stem cell properties that fat mesenchymal stem cell can be kept good.
In order to realize the purpose of the present invention, the present invention is adopted the following technical scheme that：
A kind of method of large-scale culture human adipose mesenchymal stem cells, comprises the steps：
1) fat mesenchymal stem cell culture medium is added in culture vessel, in humidity 95%, 5%CO2Incubator in Incubate in 35 DEG C and balance 30 minutes；
2) adjust fat mesenchymal stem cell culture medium temperature to 35 DEG C, pH7.05, add pretreatment microcarrier and Fat mesenchymal stem cell, the speed stirring 2h of 5RPM～20RPM；
3) 37 DEG C are warming up to, the mixing speed in 15RPM-40RPM, 5%CO2Under the conditions of cultivate 96h, period adds fat Mescenchymal stem cell culture medium.
In some embodiments, pre- described in the method for large-scale culture human adipose mesenchymal stem cells of the present invention The addition of the microcarrier of process is 0.5mg/mL-5.0mg/mL final concentration of to microcarrier.
In some embodiments, micro- described in the method for large-scale culture human adipose mesenchymal stem cells of the present invention Carrier is that Cytodex1 microcarrier, Cytodex2 microcarrier, Cytodex3 microcarrier, Cytopore microcarrier or Cytoline are micro- Carrier.In some embodiments, the method for the microcarrier pretreatment is microcarrier first with the phosphoric acid buffer immersion of pH 7.4 Bubble expansion, sterilizing；Resuspended with a small amount of culture medium after the culture medium rinse warmed using front, aseptic microcarrier.
In some embodiments, the method for the microcarrier pretreatment is microcarrier first with the phosphate buffer of pH 7.4 Soak expansion, sterilizing；Resuspended with a small amount of culture medium after the culture medium rinse warmed using front, aseptic microcarrier.
In certain embodiments, the method for the microcarrier pretreatment is dry microcarrier nothing Ca2+、Mg2+、 Expansion at least 3 hour is soaked in the phosphate buffer of pH 7.4 at room temperature, and abandoning supernatant, with new preparation nothing Ca2+、Mg2 +, pH 7.4 PBS washing microcarrier several minutes, discard PBS, change new nothing Ca2+、Mg2+, pH 7.4 PBS, then micro- load Liquid solution is sterilized with autoclaving；Before use, aseptic microcarrier is first allowed to settle, incline supernatant, then with the training of warm Foster base rinse, then allows microcarrier settle, supernatant discarded, then resuspended rear standby with a small amount of culture medium.
In some embodiments, fat described in the method for large-scale culture human adipose mesenchymal stem cells of the present invention The addition of fat mescenchymal stem cell is 0.5cells/mL-2 × 10 final concentration of to fat mesenchymal stem cell5cells/mL.
In the method for large-scale culture human adipose mesenchymal stem cells of the present invention, need in the training period to add between fat and fill Matter stem cell media.In some embodiments, the fat mesenchymal stem cell culture medium of adding is specially 48h replacing 50% culture medium.
Fat mesenchymal stem cell culture medium described in the method for large-scale culture human adipose mesenchymal stem cells of the present invention Formula be DMEM/F12+15%FBS.
The acquisition methods of fat mesenchymal stem cell of the present invention are：The human adipose mesenchymal stem cells of culture stop stirring Mix, microcarrier is settled, and supernatant discarded culture medium, with pH7.6 containing EDTA nothing Ca2+、Mg2+The PBS washing of ion, then Pancreas enzyme -EDTA solution digestion is used, after 15min, is added the cell culture fluid containing 10% (v/v) serum FBS to terminate digestion, obtains P2 is obtained 2.1 × 10 for cell6Individual cell/mL, cell quantity expand about 10 times.
Wherein, the total amount of the EDTA-PBS solution should be in (50-100) mL/g Cytodex3.
The method of large-scale culture human adipose mesenchymal stem cells of the present invention is added in culture vessel fills between fat Matter stem cell media, in humidity 95%, 5%CO2Incubator in 35 DEG C incubate balance 30 minutes；Adjustment fat mesenchymal The temperature of stem cell media to 35 DEG C, pH7.05, the microcarrier of addition pretreatment and fat mesenchymal stem cell, 5RPM～ The speed stirring 2h of 20RPM；37 DEG C are warming up to, the mixing speed in 15RPM～40RPM, 5%CO2Under the conditions of cultivate 96h, the phase Between add fat mesenchymal stem cell culture medium.Cultural method of the present invention is simple to operate, safe and effective.Experiment shows, with Existing method is compared, and after the method propagation of large-scale culture human adipose mesenchymal stem cells of the present invention, acquisition cell is more, cell Vigor and multiplication capacity by force, and can keep the form of fat mesenchymal stem cell and good stem cell properties very well, it is adaptable to The mass propgation of fat mesenchymal stem cell.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing Accompanying drawing to be used needed for technology description is had to be briefly described.
Fig. 1 shows the result figure of each surface marker of cell after 5 Flow cytometry cryopreservation resuscitation of embodiment, and wherein figure a is Cell sign CD73, figure b be CD90, figure c be CD105, figure d be CD11b, figure e for CD19, figure f for CD34, figure g be CD45, figure H is HLA-DR-A.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, Obviously, described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based in the present invention Embodiment, the every other embodiment obtained under the premise of creative work is not made by those of ordinary skill in the art, all Belong to the scope of protection of the invention.
In order to further illustrate the present invention, with reference to specific embodiment, the present invention will be described in detail.Wherein, each reality The operation that applies in example is all carried out under rigorous aseptic environment.
Embodiment 1, fat mesenchymal stem cell separation and Extraction
People's fat of fresh collection is taken, after PBS 2 times, is separated using type i collagen enzymic digestion, containing serum substitute In the culture vessel of cell culture fluid, in 37 DEG C, 5%CO2Under the conditions of quiescent culture, after 48 hours, PBS changes liquid, obtains original Fat subsitutes mescenchymal stem cell.
Wherein, the formula containing serum substitute cell culture fluid is DMEM/F12+15%FBS.
The culture vessel culture amplification of embodiment 2, fat mesenchymal stem cell
When primary fat mesenchymal stem cell covers with the 80% of plate, original fluid is discarded, washed with PBS 2 times, add The trypsase containing EDTA of ware bottom amount is covered, 1-2min is digested, period is uninterruptedly observed with inverted microscope, sees that kytoplasm is returned Contracting, space between cells increase, cell rounding, add the cell culture medium of respective volume to terminate digestion immediately, are blown with pipette repeatedly Attached cell is beaten, cell is blown and beaten, 1500r/min is centrifuged 5min, then cell culture fluid re-suspended cell is used, and according to density is 6×104Individual cell/mL, is inoculated in plate, adds fresh cell medium in 37 DEG C, 5%CO2Under the conditions of cultivate, change within 3 days Liquid, obtains P1 for cell.2 × 10 are obtained5Individual cell/mL cell, cell quantity expand about 3 times.
Embodiment 3, fat mesenchymal stem cell cultivates amplification in bioreactor containing microcarrier
1. glass blake bottle pretreatment：Working volume is cleaned up for the glass blake bottle of 500mL, is dried to completely dry Dry, silication liquid is added in bottle, slow rotating and culturing bottle makes silication immersion moisten bottle wall, after sucking silication liquid, rolling bottle is placed in logical 12h is air-dried at wind, and distilled water flushing is standby.
2. microcarrier pretreatment：Dry microcarrier Cytodex-3 is nothing Ca2+、Mg2+, pH 7.4 phosphate buffer in Expansion at least 3 hour is soaked at room temperature, and abandoning supernatant, with new preparation nothing Ca2+、Mg2+, pH 7.4 PBS wash micro- load Body several minutes, discard PBS, change new nothing Ca2+、Mg2+, pH 7.4 PBS, then microcarrier solution autoclaving go out Bacterium；Before use, first allow aseptic microcarrier settle, incline supernatant, then with the culture medium rinse of warm, then allow microcarrier Sedimentation, supernatant discarded, then resuspended rear standby with a small amount of culture medium.
3. the inoculation of fat mesenchymal stem cell：The cell culture medium of 1/3-1/2 final volume is added in glass blake bottle, Humidity 95%, 5%CO2Incubator in 35.5 DEG C incubate balance 30 minutes；The temperature of cell culture medium is adjusted to 35 DEG C, PH7.05, after the microcarrier that addition is pre-processed to concentration is for 0.5mg/mL-5.0mg/mL, is slowly added to primary fat mesenchymal and does Cell to concentration is 0.5cells/mL-2 × 105Cells/mL, is stirred under dark condition with the mixing speed of 5RPM～20RPM Mix 4 hours, temperature is all ramped up 37 DEG C within an hour afterwards, mixing speed is promoted to 15～40RPM, add cell training Foster base to final volume, in 37 DEG C, 5%CO2Under the conditions of cultivate.50% culture medium is changed per 48h,.
4. the acquisition of fat mesenchymal stem cell：Stop stirring, microcarrier is settled, and supernatant discarded culture medium, with pH7.6's Containing EDTA nothing Ca2+、Mg2+The PBS washing of ion, the total amount of EDTA-PBS solution should be in (50-100) mL/g Cytodex3.EDTA-PBS solution is discarded, is then incubated at 37 DEG C with pancreas enzyme -EDTA solution, stirs every now and then, after 15min, plus Enter the effect that the nutrient solution containing 10% (v/v) serum substitute terminates pancreatin, be obtained 2.1 × 106Individual cell/mL, cell Quantity expands about 10 times.
The freezen protective of embodiment 4, fat mesenchymal stem cell
To cover with the fat mesenchymal stem cell to 80-90% in plate, old nutrient solution is removed, with PBS 2 times, The 37 DEG C of digestion 1-2min of pancreatin being subsequently added into containing EDTA, after cell all comes off, add cell culture fluid to terminate digestion, 1500rpm/min is centrifuged 5min, removes supernatant, then cell is resuspended with stem cell cryopreserving liquid, and the fat mesenchymal of acquisition is dry thin Born of the same parents' suspension is added in cell cryopreservation tube, is placed in the program mode cooling box containing isopropanol -80 DEG C overnight, be transferred within second day - Preserved in 196 DEG C of liquid nitrogen for a long time.
Embodiment 5, flow cytometry checks recovery cell surface marker
Add after taking the recovery of 37 DEG C of 1 pipe freeze-stored cell fat mesenchymal stem cell culture medium (DMEM/F12+15%FBS) in 37 DEG C, 5%CO2Under the conditions of culture 3 days after harvest 3 × 106Cell, as flow cytometer detection.Testing result is as shown in Figure 1.Statistics inspection Survey the results are shown in Table 1.
The result of each surface marker of cell after 1 Flow cytometry cryopreservation resuscitation of table
CD73 CD90 CD105 CD11b CD19 CD34 CD45 HLA-DR
100.0% 100.0% 100.0% 1.2% 0.1% 0.2% 0.0% 0.2%
As a result show, the fat stem cell surface marker expression after cryopreservation resuscitation is normal, meets typical fat dry thin Cellular surface marker representation.

Claims (7)

1. a kind of method of large-scale culture human adipose mesenchymal stem cells, comprises the steps：
1) fat mesenchymal stem cell culture medium is added in culture vessel, in humidity 95%, 5%CO2Incubator in 35 DEG C Incubate balance 30 minutes；
2) temperature to 35 DEG C of fat mesenchymal stem cell culture medium, pH7.05, the microcarrier of addition pretreatment and fat are adjusted Mescenchymal stem cell, the speed stirring 2h of 5RPM～20RPM；
3) 37 DEG C are warming up to, the mixing speed in 15RPM-40RPM, 5%CO2Under the conditions of cultivate 96h, period adds between fat and fills Matter stem cell media.
2. method according to claim 1, the addition of the microcarrier of the pretreatment is final concentration of to microcarrier 0.5mg/mL-5.0mg/mL.
3. method according to claim 1 and 2, the microcarrier is Cytodex1 microcarrier, Cytodex2 microcarrier, Cytodex3 microcarrier, Cytopore microcarrier or Cytoline microcarrier.
4. the method according to claim 1-3 any one, the method for the microcarrier pretreatment first use pH for microcarrier 7.4 phosphate buffer soaks expansion, sterilizing；With a small amount of after the culture medium rinse warmed using front, aseptic microcarrier Culture medium is resuspended.
5. the method according to claim 1-4 any one, the addition of the fat mesenchymal stem cell be to fat Final concentration of 0.5cells/mL-2 × 10 of mescenchymal stem cell5cells/mL.
6. the method according to claim 1-5 any one, the fat mesenchymal stem cell culture medium of adding are specially 50% culture medium is changed per 48h.
7. the method according to claim 1-6 any one, the formula of the fat mesenchymal stem cell culture medium is DMEM/F12+15%FBS.
CN201611050980.1A 2016-11-24 2016-11-24 A kind of method of large-scale culture human adipose mesenchymal stem cells Pending CN106479970A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109423476A (en) * 2017-08-21 2019-03-05 青岛瑞思德生物科技有限公司 It is used to prepare the kit of fat mesenchymal stem cell
CN110117572A (en) * 2019-05-15 2019-08-13 张永国 A kind of cultural method and its culture apparatus of fat cell scale
CN111004777A (en) * 2019-12-10 2020-04-14 广东国科细胞科技有限公司 Material supplementing and culturing method for mesenchymal stem cell culture

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Publication number Priority date Publication date Assignee Title
CN102220338A (en) * 2011-05-06 2011-10-19 武汉北度生物科技有限公司 Method for expressing human 2.5 S beta-nerve growth factor in human umbilical cord mesenchymal stem cell, and separating and purifying the human 2.5 S beta-nerve growth factor
CN102321566A (en) * 2011-09-13 2012-01-18 协和干细胞基因工程有限公司 Method for extensive amplification of cell line and mesenchymal stem cell in vitro
WO2012142569A2 (en) * 2011-04-15 2012-10-18 The Regents Of The University Of California Decellularized extracellular matrix

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012142569A2 (en) * 2011-04-15 2012-10-18 The Regents Of The University Of California Decellularized extracellular matrix
CN102220338A (en) * 2011-05-06 2011-10-19 武汉北度生物科技有限公司 Method for expressing human 2.5 S beta-nerve growth factor in human umbilical cord mesenchymal stem cell, and separating and purifying the human 2.5 S beta-nerve growth factor
CN102321566A (en) * 2011-09-13 2012-01-18 协和干细胞基因工程有限公司 Method for extensive amplification of cell line and mesenchymal stem cell in vitro

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109423476A (en) * 2017-08-21 2019-03-05 青岛瑞思德生物科技有限公司 It is used to prepare the kit of fat mesenchymal stem cell
CN110117572A (en) * 2019-05-15 2019-08-13 张永国 A kind of cultural method and its culture apparatus of fat cell scale
CN111004777A (en) * 2019-12-10 2020-04-14 广东国科细胞科技有限公司 Material supplementing and culturing method for mesenchymal stem cell culture

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