CN102465112A - Human umbilical cord blood hematopoietic stem cell high-efficiency in vitro amplification technology - Google Patents
Human umbilical cord blood hematopoietic stem cell high-efficiency in vitro amplification technology Download PDFInfo
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Abstract
The invention discloses a human umbilical cord blood hematopoietic stem cell (HSC) high-efficiency in vitro amplification technology. According to the invention, umbilical cord mesenchymal stem cells (MSCs) and umbilical cord blood plasma are used in combination for carrying out HSC in vitro amplification. A process comprises steps that: umbilical cord MSCs are cultured as a trophoblast; a special culture solution containing umbilical cord blood plasma is adopted as an amplification medium of the HSCs; and umbilical cord blood karyotes are adopted as amplification initiator cells. According to the invention, exogenous cell factors are not required to be added, treatments such as mitomycin C or gamma-ray 21Gy irradiation are not required by the trophoblast, no 3-dimensional technology is required, and the HSC high-efficiency amplification can be carried out. The technology is advantaged in low cost, convenient amplification, and low product immunogenicity. The technology is suitable for large-scaled productions. The practical application of the technology plays an important role in clinical treatments and researches.
Description
Technical field
The invention belongs to biological technical field; Be specifically related to from the Cord blood of healthy subjects to extract contain hemopoietic stem cell nucleated cell as the amplification initiator cell; With the umbilical cord mesenchymal stem cells of healthy subjects as trophoderm; Be inoculated in co-cultivation in the defined medium that contains Cord blood blood plasma, thus amplifying candidate stem cell efficiently.
Background technology
Stem cell is one type and has self, the highly propagation and the cell colony of multidirectional differentiation potential, can be divided into myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell according to the differentiation potential size.(Hematopoietic stem cells HSCs) is one type of multipotential stem cell that is present in the hemopoietic tissue to hemopoietic stem cell, can directed differentiation, propagation is different blood cell lines, and further generates hemocyte.Hemopoietic stem cell is widely used in treatment white blood disease, malignant tumour, the non-pernicious disease in the blood system of severe and some heredopathia.Hemopoietic stem cell can be divided into marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell again according to the source.Compare with peripheral blood hematopoietic stem cells with marrow; Umbilical hemopoietic stem cell is more original; The self duplication ability is the strongest; And Cord blood have gather convenient, cellular immunization is immature, do not need strictness to join advantages such as type in the migration process, thereby umbilical cord blood hematopoietic stem cell has great clinical value.But the hemopoietic stem cell content of single part of bleeding of the umbilicus is low, in clinical, is difficult to the transplanting of normal type adult patient.Therefore, hemopoietic stem cell need carry out amplification in vitro before transplanting.
At present, the hematopoietic stem cell population technology mainly contains three kinds: (1) cytokine hematopoiesis support ex vivo expansion of stem cell; (2) cytotrophoblast hematopoiesis support ex vivo expansion of stem cell; (3) three-dimensional space stereoscopic culture.But there is following deficiency respectively in these three kinds of technology: also quickened the differentiation of cell when (1) cytokine promotes cell amplification, still can not reach the purpose that not only increases but also keep its stem cell function, and cytokine has cost an arm and a leg; (2) trophocyte be prone to infiltrate hemopoietic stem cell, and the hemopoietic stem cell cultivated of the part trophoderm of also can moving into, and the hemopoietic stem cell of cultivating is gathered in the crops very difficulty fully, and the allos trophocyte possibly produce the immunological rejection problem in addition; (3) three-dimensional space stereoscopic culture technical sophistication; Being difficult to carry out scale cultivates; And it mainly is the simulate bone marrow hematopoieticmicroenviron-ment that three-dimensional space is cultivated; And the original degree of marrow hemopoietic stem cells and umbilical hemopoietic stem cell and inequality, both hematopoieticmicroenviron-ments of itself are widely different, so three-dimensional space stereoscopic culture technology possibly not be the hematopoietic stem cell population that is fit to Cord blood or peripheral blood.Therefore, seek out the umbilical hemopoietic stem cell of high yield, not have immunological rejection again, and with low cost, just need to bring in constant renewal in or optimize the hematopoietic stem cell population technology.
For this reason; The present invention utilizes umbilical cord mesenchymal stem cells (Mesenchymal stem cells; MSCs) make trophoderm, the method for the adding Cord blood blood plasma umbilical cord blood hematopoietic stem cell that increases is an initiator cell with the bleeding of the umbilicus nucleated cell; Need not to add cytokine, need not the complicated 3-D technology umbilical cord blood hematopoietic stem cell that can efficiently increase.Umbilical cord mesenchymal stem cells is one type of stem cell with multidirectional differentiation potential, can produce the various kinds of cell factor, utilizes umbilical cord mesenchymal stem cells to have following advantage as trophoderm: (1) source is sufficient, draws materials conveniently, and is with low cost; (2) human umbilical cord mesenchymal stem cells content is abundant, and comparatively original, differentiation capability is strong, can separate, cultivate external, and amplification is rapid, and biological character is stable, and repeatedly going down to posterity amplification still can keep vigorous function, and competent cell source can be provided; (3) immunogenicity is extremely low, and the probability of immunological rejection is extremely low, and umbilical cord mesenchymal stem cells also has certain immune suppression function; (4) umbilical cord mesenchymal stem cells can be implanted with short by hematopoiesis support.As the ancestral cells of the main cellular constituent-stromal cell lines of hematopoieticmicroenviron-ment, mescenchymal stem cell is realized the finely regulating to hematopoiesis, the growth of hematopoiesis support stem cell through direct effect, secretory cell epimatrix and the various kinds of cell factor of cell pair cell.Mescenchymal stem cell and hemopoietic stem cell co-transplantation can promote the implantation of hemopoietic stem cell, and be significant for the transplanting succeed rate that improves hemopoietic stem cell.Simultaneously; Contain abundant hemopoieticgrowth factor in the Cord blood blood plasma; Like granulocyte colony-stimulating factor (G-CSF), monocyte G CFS (M-CSF), Hempoietine (Fpo), interleukin 1 (IL-1), interleukin 3 (IL-3), interleukin-6 (IL-6) or the like, have the hematopoietic stem cell growth of promotion and value-added functionality.Wherein some cord blood plasma factor can promote hematopoietic stem cell growth, increment with mescenchymal stem cell excretory cytokine synergy, and for example Flt3 part and EPO play a key role to the amplification of CD34+ cell.
Summary of the invention
The external efficient amplification technique that the purpose of this invention is to provide a kind of suitable umbilical cord blood hematopoietic stem cell provides competent hemopoietic stem cell for treating disease and applied research.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
In utilize normal cultured the 3rd generation, is with interior umbilical cord mesenchymal stem cells or directly utilize recovery refrigerated umbilical cord mesenchymal stem cells (also being that the 3rd generation is with interior cell), is inoculated in the DMEM/F12 nutrient solution and cultivates, and is complete when adherent to cell; It is that (this defined medium DMEM/F12: RPMI1640 is mixing in 1: 1 to defined medium that full dose is changed liquid; Other contains 10% foetal calf serum and 10% Cord blood blood plasma, pH=7.0～7.4), the nucleated cell that inoculation is extracted from Cord blood; Place co-cultivation in the incubator; Whenever changed at a distance from 3～4 days and partly measure substratum, cultivate, be expanded to the 10th～11 day, hemopoietic stem cell is the abundantest in the cell of collecting this moment.If will obtain more hemopoietic stem cell, the culturing process before repeating gets final product.
Its process is:
(1) the trophoblastic preparation of umbilical cord mesenchymal stem cells
In the 3rd generation of getting normal cultured,, (mescenchymal stem cell was from the umbilical cord of healthy subjects with interior umbilical cord mesenchymal stem cells with interior umbilical cord mesenchymal stem cells or the 3rd frozen generation of recovery; Tissue block method according to routine prepares; Normal ratio by 1: 2 goes down to posterity); Be inoculated in the DMEM/F12 nutrient solution and cultivate, adherent fully to cell.
(2) extraction of Cord blood nucleated cell
Gather the Cord blood of healthy subjects, add 5% hydroxyethylamyle (Cord blood: hydroxyethylamyle is 5: 1) in 5: 1 ratios, mixing 6～8 minutes, with 4 ℃ static 4～5 hours, get that supernatant is centrifugal can to obtain nucleated cell, it is for use to separate the blood plasma preservation that obtains.
(3) umbilical cord blood hematopoietic stem cell is cultivated, is increased
When umbilical cord mesenchymal stem cells is cultured to complete when adherent; It is that (this defined medium DMEM/F12: RPMI1640 is mixing in 1: 1 to defined medium that full dose is changed liquid; Other contains 10% foetal calf serum and 10% Cord blood blood plasma; PH=7.0～7.4), and inoculation Cord blood nucleated cell co-cultivation (Cord blood nucleated cell inoculum density is 5～8 * 10
5Cells/ml), whenever changed and partly measure substratum, got final product collecting cell to the 10th～11 day at a distance from 3～4 days.If will obtain more hemopoietic stem cell, the cell inoculation of collecting is contained in the trophoblastic nutrient solution of umbilical cord mesenchymal stem cells in what prepare again, the repetition umbilical cord blood hematopoietic stem cell is cultivated, amplification procedure gets final product.
The whole range request of crossing does not have pollutions such as bacterium, fungi, virus and mycoplasma.
Combined utilization umbilical cord mesenchymal stem cells of the present invention and cord blood plasma; Select for use the Cord blood nucleated cell as initial culturing cell; But not mononuclearcell or simple hemopoietic stem cell find that the ratio of cytodifferentiation is littler in amplification procedure, therefore; Present technique is amplifying candidate stem cell in vitro efficiently not only, but also also quickens the deficiency of breaking up when having avoided hemopoietic stem cell proliferation.
The present invention and existing hematopoietic stem cell population compared with techniques have remarkable advantages: first; Umbilical cord mesenchymal stem cells and cord blood plasma combined utilization; Efficient amplifying candidate stem cell not only; But also the differentiation that can suppress or slow down hemopoietic stem cell to a certain extent need not add the foreign cell factor, greatly reduces cost.Second; Umbilical cord mesenchymal stem cells need not processing such as ametycin or gamma-radiation 21Gy irradiation as trophoderm, because its immunogenicity is low; And has immune suppression function; Can improve the success ratio of HSCT, so even not only a spot of trophoderm mescenchymal stem cell sneak in the hemopoietic stem cell and can not produce spinoffs such as immunological rejection clinical treatment, have the promotion function on the contrary.The 3rd, the present invention has simplified cultivation, amplification procedure without the three-dimensional space technology, is convenient to large-scale production.
Embodiment
Embodiment 1, hematopoietic stem cell population
(1) preparation of umbilical cord mesenchymal stem cells
1. gather the umbilical cord of healthy subjects;
2. remove blood vessel and outer membrane in the umbilical cord;
3. umbilical cord tissue is cut into 1～2mm
3The fritter of size;
4. will shear the back fritter and plant cultivation in the petridish that is added with the DMEM/F12 nutrient solution (diameter is 100mm), put 37 ℃, 5%CO
2Cultivate in the incubator;
5. amount was changed liquid in per 3～4 days half, cultivated 14～16 days cells and grew and about 80% converge;
6. when cell about 80% converges, utilize 0.25% pancreatin pair cell to digest, with 1～3 * 10
5Cells/ml is inoculated in the cultivation of going down to posterity in the petridish;
7. when cell about 80% converges, utilize 0.25% pancreatin pair cell to digest, collect the 1st generation cell, go down to posterity by 1: 2 ratio, place 37 ℃, saturated humidity, 5%CO
2Cultivate in the incubator;
8. if need repeatedly go down to posterity, 7. repeating step gets final product;
9. if need frozen cell, as cryoprotectant, cell concn is 3～5 * 106cells/ml, carries out conventional freezing with the mixed solution of 90% foetal calf serum (FBS)+10% DMSO 99.8MIN. (DMSO).
(2) the trophoblastic preparation of umbilical cord mesenchymal stem cells
1. collected for the 3rd generation with interior (comprising for the 3rd generation) umbilical cord mesenchymal stem cells, or in the 3rd generation of refrigerated of recovering, is with interior (comprising for the 3rd generation) umbilical cord mesenchymal stem cells;
2. umbilical cord mesenchymal stem cells is inoculated in the petridish (diameter is 100mm) of DMEM/F12 nutrient solution, the adjustment cell concn is 0.4～0.6 * 10
5Cells/ml puts 37 ℃, 5%CO
2Cultivate in the incubator;
3. adherent fully to umbilical cord mesenchymal stem cells, need not processing such as ametycin or gamma-radiation 21Gy irradiation.
(3) extraction of Cord blood nucleated cell
Gather the Cord blood of healthy subjects, be stored in the anticoagulation bag, add 5% hydroxyethylamyle (HES) (Cord blood: hydroxyethylamyle is 5: 1) by 5: 1 volume ratios; Mix, left standstill 4～5 hours sedimented red cell in 4 ℃ of refrigerators; Get supernatant; The centrifugal 10min of 2000rpm obtains nucleated cell, wherein contains 0.83%CD34+ cell (CD34 is a hemopoietic stem cell surface antigen).
(4) umbilical cord blood hematopoietic stem cell is cultivated, is increased
The trophoderm mescenchymal stem cell is cultured to complete when adherent; It is that (this defined medium DMEM/F12: RPMI1640 is mixing in 1: 1 to defined medium that full dose is changed liquid; Other contains 10%FBS and 10% Cord blood blood plasma; PH=7.0～7.4), and inoculation Cord blood nucleated cell co-cultivation (Cord blood nucleated cell inoculum density is 5～8 * 10
5Cells/ml), whenever changed at a distance from 3～4 days and partly measure substratum, got final product collecting cell to the 10th～11 day, hemopoietic stem cell is the abundantest in the cell of collecting this moment.If will obtain more hemopoietic stem cell; The cell inoculation of collecting is contained in the trophoblastic defined medium of umbilical cord mesenchymal stem cells in what prepare again; Whenever changed at a distance from 34 days and partly measure substratum; Get final product collecting cell to the 10th～11 day, repeatedly repeat this cultivation, amplification procedure can obtain a large amount of hemopoietic stem cells.
Embodiment 2, hematopoietic stem cell population effect measuring
(1) measures grouping
The Cord blood nucleated cell that extracts is inoculated in following each group, to compare the hematopoietic stem cell population effect.
A organizes (only containing MSC): umbilical cord mesenchymal stem cells is made trophoderm, and substratum is: DMEM/F12: RPMI1640 is mixing in 1: 1, and other contains 10%FBS, pH=7.0～7.4;
B organizes (only factor-containing): no umbilical cord mesenchymal stem cells trophoderm; Substratum is: DMEM/F12: RPMI1640 is mixing in 1: 1; Other contains 10%FBS and stem cell factor (SCF) 30ng/ml, TSF (TPO) 20ng/ml, FLT-3 part (FL) 20ng/ml, pH=7.0～7.4;
C organizes (MSC+ cytokine): umbilical cord mesenchymal stem cells is made trophoderm; Substratum is: DMEM/F12: RPMI1640 is mixing in 1: 1; Other contains 10%FBS and stem cell factor (SCF) 30ng/ml, TSF (TPO) 20ng/ml, FLT-3 part (FL) 20ng/ml, pH=7.0～7.4;
D organizes (MSC+ cord blood plasma): umbilical cord mesenchymal stem cells is made trophoderm, and substratum is: DMEM/F12: RPMI1640 is mixing in 1: 1, and other contains 10%FBS and 10% Cord blood blood plasma, pH=7.0～7.4.
(2) expanding effect is measured
More than each group respectively at cultivating the 6th and the 11st day mensuration CD34+ cell and colony forming unit (Colony forming unit, CFU) amplification times.CD34 is a hemopoietic stem cell surface antigen; The propagation situation that how much reflects hemopoietic stem cell of CD34+ cell count; The CD34+ cell adopts MiniMACS immune magnetic adsorption column tripping device to separate, and utilizes FACS Calibur flow cytometry analysis CD34+ cell purity; Colony forming unit (CFU) comprising: grain monosystem colony forming unit (Colony forming unit for granulocytes/macrophage; CFU-GM), grain clearance permit macronucleus colony forming unit (Colony forming unit for granulocytes; Erythroid; Monocytes and megarkaryocytes; CFU-GEMM), BFU-E (Burst forming unit erythroid; BFU-E) and high value-added potential colony forming unit (High proliferative colony forming unit HPP-CFU), mainly reflects the proliferation and differentiation situation of hemopoietic stem cell; Utilize methylcellulose gum substratum (including stem cell factor, interleukin-3, interleukin-6, granulocyte colony-stimulating factor, the unicellular G CFS of grain and TSF) culture assays, colony with cell count greater than 50 countings.
A organizes (only containing MSC): it is 4.1 times and 6.0 times that CD34+ cell and colony forming cell (CFU) amplification times the 6th day was 2.0 times and 4.1 times, the 11st day;
B organizes (only factor-containing): it is 3.5 times and 6.5 times that CD34+ cell and colony forming cell (CFU) amplification times the 6th day was 3.0 times and 4.3 times, the 11st day;
C organizes (MSC+ cytokine): it is 16.8 times and 10.2 times that CD34+ cell and colony forming cell (CFU) amplification times the 6th day was 4.4 times and 6.3 times, the 11st day;
D organizes (MSC+ cord blood plasma): it is 46.6 times and 22.7 times that CD34+ cell and colony forming cell (CFU) amplification times the 6th day was 20.7 times and 11.8 times, the 11st day.
Find through comparative study: combined utilization umbilical cord mesenchymal stem cells and Cord blood blood plasma; With the Cord blood nucleated cell is initiator cell; Amplifying candidate stem cell in vitro efficiently; And this method has certain inhibition to the differentiation of hemopoietic stem cell or delays slow effect, therefore utilizes this technology can be for clinical treatment and research provide with low cost, amplification is easy, the hemopoietic stem cell of reduced immunogenicity.
Claims (10)
1. the external efficient amplification technique of people source umbilical cord blood hematopoietic stem cell; It is characterized in that combined utilization umbilical cord mesenchymal stem cells and cord blood plasma; With the Cord blood nucleated cell is that initiator cell carries out amplification in vitro, and its process is defined medium → inoculation Cord blood nucleated cell → cultivation of containing cord blood plasma, increases as trophoderm → change liquid to adherent fully for cultivating umbilical cord mesenchymal stem cells.
2. amplification technique according to claim 1 is characterized in that combined utilization human umbilical cord mesenchymal stem cells and people's cord blood plasma.
3. amplification technique according to claim 2 is characterized in that human umbilical cord mesenchymal stem cells is the cell of the 3rd generation with interior (comprising for the 3rd generation).
4. amplification technique according to claim 1 is characterized in that cultivating umbilical cord mesenchymal stem cells to fully adherent as trophoderm.
5. amplification technique according to claim 1, used defined medium contains DMEM/F12, RPMI1640, FBS and Cord blood blood plasma in it is characterized in that cultivating.
6. amplification technique according to claim 5 is characterized in that DMEM/F12 mixes by 1: 1 with RPMI1640 in the defined medium.
7. amplification technique according to claim 5 is characterized in that containing in the defined medium Cord blood blood plasma of 10% volumetric concentration, but is not limited thereto concentration.
8. amplification technique according to claim 1 is characterized in that amplification procedure is every partly to measure substratum at a distance from replacing in 3～4 days.
9. amplification technique according to claim 1 is characterized in that with the human cord blood nucleated cell as the amplification initiator cell.
10. amplification technique according to claim 1 is characterized in that single cultivation, amplification cycle are 10～11 days, but are not limited thereto the cycle.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103330720A (en) * | 2013-07-18 | 2013-10-02 | 广州市天河诺亚生物工程有限公司 | Mixing stem cell injection and preparation method thereof |
| CN103585179A (en) * | 2013-10-11 | 2014-02-19 | 中国人民解放军第三军医大学第一附属医院 | Pharmaceutical composition and application thereof in preparation of drug for treating bone marrow hematopoietic function disorder |
| CN105713879A (en) * | 2014-12-01 | 2016-06-29 | 顺昊细胞生物技术（天津）股份有限公司 | Culture system for umbilical cord blood hematopoietic stem cell amplification and application thereof |
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| CN108165531A (en) * | 2017-12-23 | 2018-06-15 | 淮北智淮科技有限公司 | A kind of Cord blood mononuclear cells cultural method of candidate stem cell |
| CN110951687A (en) * | 2019-12-30 | 2020-04-03 | 贵州泛特尔细胞生物技术有限公司 | Method for amplifying placenta source hemopoietic stem cells |
| CN111019894A (en) * | 2013-05-20 | 2020-04-17 | 位于西奈山的伊坎医学院 | Enriched and expanded human cord blood stem cells for the treatment of hematological disorders |
| CN112481207A (en) * | 2020-11-27 | 2021-03-12 | 北京广未生物科技有限公司 | Method for promoting cord blood hematopoietic stem cell proliferation by using adipose-derived stem cells |
| CN113750220A (en) * | 2020-06-02 | 2021-12-07 | 南京大学 | Application of mesenchymal stem cell combined TPO and analogue thereof in treating chronic myelogenous leukemia |
| CN115449510A (en) * | 2022-10-26 | 2022-12-09 | 银丰生物工程集团有限公司 | Colony culture medium without exogenous factor addition and preparation method and application thereof |
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| CN111019894A (en) * | 2013-05-20 | 2020-04-17 | 位于西奈山的伊坎医学院 | Enriched and expanded human cord blood stem cells for the treatment of hematological disorders |
| CN103330720B (en) * | 2013-07-18 | 2014-10-01 | 广州市天河诺亚生物工程有限公司 | Mixing stem cell injection and preparation method thereof |
| CN103330720A (en) * | 2013-07-18 | 2013-10-02 | 广州市天河诺亚生物工程有限公司 | Mixing stem cell injection and preparation method thereof |
| CN103585179A (en) * | 2013-10-11 | 2014-02-19 | 中国人民解放军第三军医大学第一附属医院 | Pharmaceutical composition and application thereof in preparation of drug for treating bone marrow hematopoietic function disorder |
| CN105713879A (en) * | 2014-12-01 | 2016-06-29 | 顺昊细胞生物技术（天津）股份有限公司 | Culture system for umbilical cord blood hematopoietic stem cell amplification and application thereof |
| CN105713879B (en) * | 2014-12-01 | 2020-05-08 | 顺昊细胞生物技术（天津）股份有限公司 | Culture system for umbilical cord blood hematopoietic stem cell amplification and application thereof |
| CN106801038A (en) * | 2015-11-26 | 2017-06-06 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of utilization Three-dimensional cell culture system promotes the cell culture processes of umbilical cord blood hematopoietic stem cell fast and stable propagation |
| CN107446890A (en) * | 2017-08-29 | 2017-12-08 | 云南舜喜再生医学工程有限公司 | A kind of method of amplification in vitro umbilical cord blood hematopoietic stem cell |
| CN108165531A (en) * | 2017-12-23 | 2018-06-15 | 淮北智淮科技有限公司 | A kind of Cord blood mononuclear cells cultural method of candidate stem cell |
| CN110951687A (en) * | 2019-12-30 | 2020-04-03 | 贵州泛特尔细胞生物技术有限公司 | Method for amplifying placenta source hemopoietic stem cells |
| CN110951687B (en) * | 2019-12-30 | 2021-08-17 | 贵州泛特尔细胞生物技术有限公司 | Method for amplifying placenta source hemopoietic stem cells |
| CN113750220A (en) * | 2020-06-02 | 2021-12-07 | 南京大学 | Application of mesenchymal stem cell combined TPO and analogue thereof in treating chronic myelogenous leukemia |
| CN113750220B (en) * | 2020-06-02 | 2023-11-03 | 南京大学 | Application of mesenchymal stem cells combined with TPO and analogues thereof in treatment of chronic myelogenous leukemia |
| CN112481207A (en) * | 2020-11-27 | 2021-03-12 | 北京广未生物科技有限公司 | Method for promoting cord blood hematopoietic stem cell proliferation by using adipose-derived stem cells |
| CN112481207B (en) * | 2020-11-27 | 2021-10-12 | 广州市拜沃思生物科技有限公司 | Method for promoting cord blood hematopoietic stem cell proliferation by using adipose-derived stem cells |
| CN115449510A (en) * | 2022-10-26 | 2022-12-09 | 银丰生物工程集团有限公司 | Colony culture medium without exogenous factor addition and preparation method and application thereof |
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