CN104419659A - Cultivation and mass-production method of adipose-derived stem cells and stem cell secretion of adipose-derived stem cells - Google Patents
Cultivation and mass-production method of adipose-derived stem cells and stem cell secretion of adipose-derived stem cells Download PDFInfo
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Abstract
The invention discloses a cultivation and mass-production method of adipose-derived stem cells and stem cell secretion of the adipose-derived stem cells. The method comprises the following steps: (A) collecting an adipose cell sample; (B) separating adipose-derived stem cells from the adipose cell sample; (C) planting the adipose-derived stem cells into a three-dimensional carrier, and cultivating in a bioreactor; and (D) collecting stem cell secretion which is secreted by the adipose-derived stem cells, wherein the stem cell secretion at least comprises one of a vascular endothelial growth factor, a hepatocyte growth factor, an interleukin-6 and type 1 collagen, or combination thereof, and therefore, heavy and complicated steps, and economic cost such as manpower and equipment can be removed, adipose-derived stem cells can be quickly and massively cultivated, the stem cell secretion of the adipose-derived stem cells can be produced, and the cultivation and mass-production method can be applied to the fields of disease treatment, cytothesis and regenerative medicine in the future.
Description
Technical field
The present invention relates to cultivation and the mass production method of a kind of stem cell and stem cell secretion thing thereof, particularly relate to a kind of cultivation of fat stem cell and the method for its stem cell secretion thing of volume production.
Background technology
Flourish based on biotechnology in recent years and medical skill, stem cell, because of its distinctive differentiation, hyperplasia and tissue repair potential, makes it increasingly important in Bio-pharmaceutical Industry.It is no matter the significantly growth from global stem cell associated companies quantity, market value, or united States food and drug administration (FDA) accepts with situations such as the risings year by year of the clinical case of stem cell application, all demonstrating stem-cell therapy has become international bio scientific and technological industry and has given priority to project.
But the quick differentiation capability of stem cell often makes it after the 3 to 4 subculture, lose its stem cell properties gradually, so that subsequent experimental often cannot meet clinical expection.Even the stem cell of first culture, also different from clinical manifestation with its characteristic of the stem cell of culture-based method.In addition, be different from the detection bodies sources such as bone marrow stem cell, cord blood stem cell and embryonic stem cell and obtain and be comparatively not easy, fat obtains source for relatively easy detection bodies, and detection bodies to obtain the sense of discomfort that process produces contributor light and slow compared with other sources.
In addition, except the medical applications of stem cell itself, more and more research for stem cell secretion thing shows, and the compound composing factor of stem cell secretion thing also has good behaviour for regenerative medicine.America and Europe all has research report, cultivates impaired liver cell or other mammal tissues, all have the achievement of hyperplasia and tissue repair with stem cell secretion thing.
Therefore, namely the primary and foremost purpose of research is to delay differentiation of stem cells, maintains its stem cell properties and simultaneously mass propgation stem cell and stem cell secretion thing thereof, in order to follow-up study present stage.How by improvement conventional dry cell culture processes, and then manufacture and produce a large amount of stem cell and its stem cell secretion thing to meet the demand of cell biological educational circles instantly, become a problem urgently to be resolved hurrily.
Summary of the invention
Based on above-mentioned known problem and demand, the present invention mainly delays cytodifferentiation with the innovation 3D culture method being different from conventional cell training method and maintains stem cell properties, and the perfusion system of collocation timing is simultaneously to stimulate stem cell secretion produce raw.Due to the impact that 3D steric environment culture method divides the maintenance of stem cell properties and the composition of its secretory product, all can be applicable to regenerative medicine and as the development potentiality of other medical fields.Therefore the present invention makes the demand of producing a large amount of stem cell and its stem cell secretion thing and meeting cell biological educational circles instantly with stereoscopic culture legal system.
One object of the present invention is cultivated and the method for its stem cell secretion thing of volume production for providing a kind of fat stem cell, utilizes the 3D culture technique large scale cultivating fat stem cell of innovation research and development and volume production collect its stem cell secretion thing and go out individualized stem cell products in order to later stage researchdevelopment.
The step of the cultural method of above-mentioned fat stem cell comprises: (A) collects adipocyte sample; (B) fat stem cell in this adipocyte sample is separated; (C) this fat stem cell is distributed on three-dimensional carrier with dry type cell seeding method, and is placed in thing reactor in all one's life, so that nutrient solution perfusion and the jejunitas method of cell timing are cultivated at regular time and quantity; (E) the stem cell secretion thing secreted by this fat stem cell is collected; Wherein this stem cell secretion thing at least comprises vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), pHGF (Hepatocyte growth factor, HGF), be situated between white element-6 (Interleukin-6, IL-6), 1 collagen type (collagen type I) one of them or its combination.
In the above-mentioned methods, preferably, step (C) comprises so that nutrient solution perfusion and the jejunitas method of cell timing are cultivated at regular time and quantity further; The number of the three-dimensional carrier that each bio-reactor loads is between 100 ~ 1500; Cultivating the fat stem cell total quantity obtained is 1 × 10
8~ 1 × 10
9individual/culturing bottle.The concentration of the vascular endothelial growth factor of stem cell secretion thing can can can between 0 ~ 42ng/ml between the concentration of 0 ~ 523pg/ml, 1 collagen type between the concentration of 0 ~ 1780pg/ml, the white element-6 that is situated between the concentration of 0 ~ 2090pg/ml, pHGF,, and the paste rate of fat stem cell on three-dimensional carrier preferably can reach 90%.
The present invention has following advantage relative to conventional cell culture method:
By the method for dimensional culture fat stem cell, complicated operation steps and manpower can be saved, reduce the device space, volume production stem cell secretion thing and save the Financial cost of cell culture fluid.In addition, dry type cell implanted prosthetics can be improved 3D culture method cell in the past and paste the poor bottleneck of rate and with nutrient solution perfusion at regular time and quantity and the jejunitas method of cell timing, change composition and the turnout of stem cell secretion thing, and cultivation consumptive material consumption and the expenditure of 8 to 10 times can be saved in economic benefit.By disclosure of the present invention, fat stem cell and a large amount of its stem cell secretion thing of production can be cultivated by rapid, high volume, apply to provide the research in the fields such as disease treatment, cytothesis and regenerative medicine.The research and development that the cell culture system of original creation also can be cultivated as other cell types are applied.
Accompanying drawing explanation
Fig. 1 is the flow chart of steps of the cultural method showing fat stem cell of the present invention;
Fig. 2 is the purification step schema of fat stem cell;
Fig. 3 is the cultivation of fat stem cell and the production stage schema of stem cell secretion thing;
Fig. 4 shows the fat stem cell survival rate after purified cultivation;
Cell enchylema to be uniformly distributed in the three-dimensional carrier in culturing bottle by Fig. 5 display with dry type cell seeding method after pastes rate;
Fig. 6 shows the glucose content comparative result in method of the present invention and known nutrient solution;
The concentration that Fig. 7 A ~ 7D shows various stem cell secretion thing compares.
Embodiment
Consult Fig. 1, it is the flow chart of steps of the method for display fat stem cell cultivation of the present invention and its stem cell secretion thing of volume production.The present invention mainly provides cultivation and the mass production method of a kind of fat stem cell and stem cell secretion thing thereof, and step comprises: collect adipocyte sample (step 101); Be separated the fat stem cell (step 102) in this adipocyte sample; This fat stem cell is distributed on three-dimensional carrier with dry type cell seeding method, and is placed in all one's life thing reactor and carries out cultivating (step 103); Collect the stem cell secretion thing (step 104) secreted by this fat stem cell in this nutrient solution.Wherein this stem cell secretion thing at least comprises vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), pHGF (Hepatocytegrowth factor, HGF), be situated between white element-6 (Interleukin-6, IL-6), 1 collagen type (collagen typeI) one of them or its combination.
In the above-mentioned methods, preferably, the concentration of the vascular endothelial growth factor of stem cell secretion thing is between 0 ~ 2090pg/ml, the concentration of pHGF is between 0 ~ 1780pg/ml, the concentration of the white concentration of plain-6 that is situated between 0 ~ 523pg/ml, 1 collagen type between 0 ~ 42ng/ml, and cultivating the fat stem cell total quantity obtained is 1 × 10
8~ 1 × 10
9individual/culturing bottle, and the paste rate of fat stem cell on three-dimensional carrier preferably can reach 90%, the number of the three-dimensional carrier that each bio-reactor loads is between 100 ~ 1500.Foregoing is only the leading each step of summary, embodiment will describe in detail as after.
The separation of fat stem cell and purifying
Consult Fig. 2, the purification step schema of its display fat stem cell.As shown in the figure, step 201 ~ 208 are respectively: cleaning fat, below watery blood waste liquid (step 201) is removed after fatty layering, fat is moved to centrifugal (step 202) in centrifuge tube, fat lump is poured into after shredding in dish and move to (step 203) in centrifuge tube, incubator is put into after adding Collagenase, and slow rotation is to mix (step 204), mixed solution is filtered to centrifugal (step 205) in centrifuge tube, centrifugal rear taking-up cell precipitation thing cleaning, and then centrifugal (step 206), cell precipitation thing is disperseed with dPBS, and then filter centrifugation (step 207), get centrifugal supernatant liquor and send inspection department to carry out steriling test, micro-slurry bacterium and endotoxin content test (step 208), the fat stem cell of centrifugal rear gained is cultivated in conventional flask again.Fatty detection bodies due to step 201 ~ 208 is collected and stem cell is separated identical with known technology, is not described at this for detail section.
The collection of high-effect fat stem cell cultivation and stem cell secretion thing thereof
After the separation completing above-mentioned fat stem cell and purification step, first carry out first time amplification culture, its survival rate as shown in Figure 4.Then carry out follow-up high-effect fat stem cell cultivation and the collection of stem cell secretion thing thereof, it is respectively step 301 ~ 310, as shown in Figure 3.
By the cell suspension in culture plate (step 301) in container.
With dry type cell seeding method enchylema is uniformly distributed on the three-dimensional carrier in culturing bottle (step 302).Three-dimensional carrier quantity is preferably 100 ~ 1,500, its material can be glass fibre, PET or other there is hole, hole, and be applicable to adipocyte and paste cultivation, have the material of bio-compatibility concurrently.Known wet type cell seeding method is that three-dimensional carrier, nutrient solution and cell are placed in centrifuge tube, allows cell be attached on carrier voluntarily, but this method need leave standstill and puts and need regularly rock, and step is more loaded down with trivial details and required time is longer.And be in this dry type cell seeding method adopted and known wet type cell seeding method difference, dry type cell seeding method is directly distributed in by the enchylema containing specific quantity on dry three-dimensional carrier, save above-mentioned leaving standstill to put, step and the required time such as to rock, and significantly improve cell through the dry type planting method of this improvement paste rate on carrier.
Consult Fig. 5, after about 200 minutes, paste rate with the cell of dry type cell seeding method gained and can reach about 90%, positively improve known fat stem cell culture technique and paste the not high shortcoming of rate.The step of dry type cell seeding method is as follows:
Previous operations
A. with cell culture technology in conventional flask by fat stem cell amplification culture.
B. 100 ~ 1500 three-dimensional carriers are prepared before operation.
Operation steps
1. remove the old nutrient solution in culture plate.2. clean cell 2 times with dPBS (Du's formula phosphoric acid buffer).3. add appropriate trypsin-EDTA solutions, all cells is suspended.4. with appropriate nutrient solution by cell suspending liquid and rear centrifugal 300-500g/3-10 minute.5. remove supernatant liquor, retain pipe floor cells, cell broken up with nutrient solution 500 ten thousand cells that are suspended into every milliliter about 1,000 ten thousand to 1 1000.6. cell suspending liquid is uniformly distributed on the three-dimensional carrier in bio-reactor special-purpose bottle.This step is preferably and uses above-mentioned dry type cell seeding method, can obtain good cell and paste rate.7. bio-reactor special-purpose bottle is placed in 37 DEG C, 5%CO
260-120 minute (step 303) is left standstill in incubator.
Nutrient solution is poured in bio-reactor special-purpose bottle to be placed in incubator and is cultivated (step 304).Nutrient solution composition comprises nutrient solution (any one such as DMEM, DMEM/F12, F12, StemPro...), foetal calf serum (FBS), microbiotic (gentamicin, penicillin as used herein ... Deng), similar to known culture dish method, do not repeat them here.Just using the additive containing animal-origin when carrying out cell implantation and cell number amplifies, such as FBS, must be the source of CTS grade or mad cow syndrome Pest-or disease-free area.
Serial connection bio-reactor special-purpose bottle and the serum bottle (step 305) containing nutrient solution.
Again by traditional thread binding for serises connecting tube on peristaltic pump (step 306).
Perfusion cell culture fluid (step 307) at regular time and quantity.Can coordinate in this step and give the jejunitas of specific times and specified time length, with the proportion of composing and the output that stimulate stem cell to adjust its stem cell secretion thing.
Cultivate the stem cell secretion thing (step 308) can collected after tens of hours containing high density somatomedin.When carrying out the collection of stem cell secretion thing, can not animal serum be used, carry out with the nutrient solution of animal origin-free (xeno-free), antibiotic-free or basic culture solution, to avoid because cause containing non-human components user irritated in finished product.Below cultivate in bio-reactor for fat stem cell and collect the detailed step of stem cell secretion thing, to make above-mentioned steps more clear and definite:
1. be incubated on three-dimensional carrier by fat stem cell, cultivate in bio-reactor, nutrient solution is the DMEM/F12 containing 10% foetal calf serum.
2. when cultivating, bio-reactor special-purpose bottle is placed on bio-reactor.
3. between incubation period every 2-3 days, change nutrient solution with buffer solution for cleaning.Damping fluid can be dPBS or PBS.
4. cultivate after 7 ~ 10 days and can start to collect fat stem cell secretory product.
5. the bio-reactor special-purpose bottle of three-dimensional carrier after removing old nutrient solution, is contained with about 1-2 litre buffer solution for cleaning.Damping fluid can be dPBS or PBS.
6. add nutrient solution DMEM/F12.The nutrient solution used can be any one such as DMEM, DMEM/F12, F12, StemPro..., is not limited only to DMEM/F12 described herein.
7. with perfusion pipeline series winding serum bottle and bio-reactor special-purpose bottle, and carry peristaltic pump to regulate and control flow velocity and flow, reach thus at regular time and quantity and object that cell is regularly jejunitas.
8. cultivate the fat stem cell secretory product in 48 ~ 72 h before harvest cell culture fluids.
Cell can proceed to cultivate and collect stem cell secretion thing (step 309).
The stem cell secretion thing collected preserves (step 310) with fixed temperature and humidity.
With regard to the known cultural method utilizing culture dish, the human adipose stem cell that 10cm culture dish can be cultivated is about 1 × 10
6individual, the nutrient solution volume that can obtain is about 10 ~ 15ml; The human adipose stem cell that T-175 cultivation angle bottle can be cultivated is about 2.5 × 10
6individual, the nutrient solution volume that can obtain is about 40 ~ 100ml, and the fat stem cell total quantity of present method is better reaches 1 × 10
9individual/culturing bottle, and the cell culture fluid that once can obtain mostly is 12L most.
Cell differentiation compares
Consult Fig. 6, it shows the glucose content comparative result in method of the present invention and known nutrient solution.Because past research finds that stem cell is relevant with cell differentiation to glucose utilization rate, when cell differentiation is larger, higher to the utilization ratio of glucose, can be found by glucose content in the nutrient solution of analysis conventional method and dimensional culture method of the present invention, the nutrient solution glucose content of dimensional culture method is significantly higher than Traditional Method, shows that fat stem cell cultivates the characteristic compared with maintaining stem cell under three-dimensional environment.
Stem cell secretion substrate concentration compares
The stem cell secretion thing obtained is collected through above-mentioned steps, which includes multiple stem cell secretion thing, wherein at least include vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), pHGF (Hepatocyte growth factor, HGF), be situated between white element-6 (Interleukin-6, IL-6), 1 collagen type (collagen type I).Except above-mentioned several factor, the stem cell cultivating gained also may containing other various kinds of cell hormones (cytokine) and extracellular matrix (ECM); Such as: somatomedin (GrowthFactor), cytokine (Interlukin) and cytoskeleton (cytoskeleton) etc., this only with vascular endothelial growth factor, pHGF, the white element of Jie-6,1 collagen type be detected as example, be not limited only to this.
The result of the inventive method and traditional culture plate is made comparisons, using the concentration of the above-mentioned factor as benchmark.Refer to Fig. 7 A ~ 7D, and consult table 1 simultaneously.Innovation method is cultural method disclosed by the invention, when cultivating, culturing bottle includes 1000 three-dimensional carriers, black, white, grey bar (bar) represents respectively to be collected after 48 ~ 50 hours with 500mL, 2700mL and 6000mL nutrient solution perfusion, and detects VEGF, HGF, LI-6 and Collagen type I concentration respectively with ELISA kit.Traditional Method, for using T75 culturing bottle, is collected after cultivating 48 ~ 50 hours, and detect VEGF, HGF, LI-6 and Collagen type I concentration respectively with ELISA kit with 34mL DMEM/F12 nutrient solution.
Under the cultivation situation of same cell source, nutrient solution condition, temperature and gas concentration lwevel, VEGF concentration measured in the nutrient solution obtained with T75 culturing bottle is 246.78 ± 58.64pg/ml, and cultural method of the present invention is 1987.18 ± 103.25pg/ml.HGF concentration measured in the nutrient solution obtained with T75 culturing bottle is 299.31 ± 86.3pg/ml, and cultural method of the present invention is 1613.25 ± 166.71pg/ml.IL-6 concentration measured in the nutrient solution obtained with T75 culturing bottle is 76.21 ± 22.65pg/ml, and cultural method of the present invention is 514.12 ± 9.29pg/ml.Collagen type I concentration measured in the nutrient solution obtained with T75 culturing bottle is 17.75 ± 6.96ng/ml, and cultural method of the present invention is 39.45 ± 2.55ng/ml.VEGF, HGF, IL-6 and collagen type I concentration of two kinds of training methods carries out two tail statistical study with Student ' s t test, result display all has significant difference, therefore uses VEGF, HGF, IL-6 and collagen type I concentration measured by method of the present invention significantly higher.
Table 1
| Traditional Method | Innovation method (the present invention) | |
| VEGF(pg/mL) | 246.78±58.64 | 1987.18±103.25 |
| HGF(pg/mL) | 299.31±86.3 | 1613.25±166.71 |
| IL-6(pg/mL) | 76.21±22.65 | 514.12±9.29 |
| collagen type I(ng/mL) | 17.75±6.96 | 39.45±2.55 |
In sum, the cultural method that the present invention discloses has following advantage relative to conventional cell culture method:
Short-term mass propgation fat stem cell, save complicated operation steps and manpower, reduce the device space, volume production stem cell secretion thing and save the Financial cost of cell culture fluid.In addition, there is dry type cell implanted prosthetics can improve known culture method cell and paste the poor bottleneck of rate and by nutrient solution perfusion at regular time and quantity and cell regularly jejunitas method, to change composition and the turnout of stem cell secretion thing.
For T175 Tissue Culture Flask, educable area is 175cm
2, each operation only can operate single Tissue Culture Flask.Effective culture area of the three-dimensional carrier that the present invention uses is about 25cm
2, and each bio-reactor can load at most 1,500 carriers altogether only needs 500ml nutrient solution, be equivalent to cultivate 142 to 214 T175 Tissue Culture Flasks, this needs about 4,000-6,500ml nutrient solutions simultaneously.Cultivation consumptive material consumption and the expenditure of 8 to 10 times can be saved in economic benefit aspect.Cultivate batch (a whole cell culture incubator) for one, a cell culture incubator can cultivate 144 T25 Tissue Culture Flasks or 2 bio-reactors simultaneously.If cultivated with T25 Tissue Culture Flask and the high-effect system of 3D of the present invention respectively, compared with the traditional mode of production mode utilizing T25 Tissue Culture Flask, each cultivation batch of the high-effect system of 3D can be reduced work hours at least 24 hours, reaches the output of about 27 times simultaneously.
The detection of stem cell secretion thing
For verifying the stem cell secretion thing produced, the impacts such as abnormal hyperplasia can not be produced on cell, by the nutrient solution collected, carrying out mankind's fibroblast (human foreskin fibroblast, HSF) and cultivating.
Cultivate and carry out the analysis of cell proliferation multiplying power after 3 days, with 20% or 100% stem cell secretion thing cultured cells, cell proliferation multiplying power is respectively 1.7 ± 0.3 and 1.3 ± 0.2.
4.4 ± 0.2 and 6.3 ± 0.4 are respectively with the HSF cell proliferation multiplying power that 5% or 10%FBS are cultivated 3 days, all remarkable in stem cell secretion object height.Above-mentioned test proves, the security concerns that the stem cell secretion thing that cultural method of the present invention is produced does not have human body to use.
According to foregoing, the present invention can cultivate fat stem cell and its stem cell secretion thing of volume production by rapid, high volume, applies to provide the research in the fields such as disease treatment, cytothesis and regenerative medicine.The cell culture system of original creation also can be used as the research and development application of other cell types cultivation.
As seen from the above embodiment; the cultural method of fat stem cell provided by the present invention and stem cell secretion thing thereof have the utility value in industry really; above describes the explanation being only the preferred embodiments of the present invention; those skilled in the art can make the improvement of other kind according to above-mentioned explanation, these change and are also contained in scope of patent protection that spirit of the present invention and claim limit.
Claims (8)
1. a method for fat stem cell cultivation and its stem cell secretion thing of volume production, its step at least comprises:
(A) adipocyte sample is collected;
(B) fat stem cell in this adipocyte sample is separated;
(C) this fat stem cell is distributed on three-dimensional carrier with dry type cell seeding method, and the nutrient solution being placed in thing reactor in all one's life is cultivated;
(D) the stem cell secretion thing secreted by this fat stem cell is collected;
Wherein this stem cell secretion thing at least comprises vascular endothelial growth factor (Vascular endothelialgrowth factor, VEGF), pHGF (Hepatocyte growth factor, HGF), be situated between white element-6 (Interleukin-6, IL-6), 1 collagen type (collagen type I) one of them or its combination.
2. the method for claim 1, wherein step (C) comprises further so that nutrient solution perfusion and the jejunitas method of cell timing are cultivated at regular time and quantity.
3. the method for claim 1, wherein the concentration of the vascular endothelial growth factor of this stem cell secretion thing is between 0 ~ 2090pg/ml, the concentration of pHGF between the concentration of 0 ~ 1780pg/ml, the white element-6 that is situated between the concentration of 0 ~ 523pg/ml, 1 collagen type between 0 ~ 42ng/ml.
4. the method for claim 1, this fat stem cell total quantity of wherein cultivating acquisition is 1 × 10
8~ 1 × 10
9individual/culturing bottle.
5. the method for claim 1, wherein the paste rate of this fat stem cell on three-dimensional carrier is 90%.
6. the method for claim 1, wherein the number of the three-dimensional carrier of this each bio-reactor loading is between 100 ~ 1500.
7. a stem cell secretion thing, with acquired by the method as described in claim 1, wherein this stem cell secretion thing at least comprises vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), pHGF (Hepatocyte growth factor, HGF), be situated between white element-6 (Interleukin-6, IL-6), 1 collagen type (collagen type I) one of them or its combination.
8. stem cell secretion thing as claimed in claim 7, wherein the concentration of the vascular endothelial growth factor of this stem cell secretion thing is between 0 ~ 2090pg/ml, the concentration of pHGF between the concentration of 0 ~ 1780pg/ml, the white element-6 that is situated between the concentration of 0 ~ 523pg/ml, 1 collagen type between 0 ~ 42ng/ml.
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| CN110066726A (en) * | 2019-04-29 | 2019-07-30 | 广州迈普再生医学科技股份有限公司 | Fully automatic system and its cell culture processes for the continuous dimensional culture of cell |
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| CN113151158A (en) * | 2021-05-19 | 2021-07-23 | 杭州憶盛医疗科技有限公司 | Tissue stem cell separation and function evaluation system |
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