CN105018428A - In-vitro amplification method for cord blood hematopoietic stem cells - Google Patents
In-vitro amplification method for cord blood hematopoietic stem cells Download PDFInfo
- Publication number
- CN105018428A CN105018428A CN201510277206.3A CN201510277206A CN105018428A CN 105018428 A CN105018428 A CN 105018428A CN 201510277206 A CN201510277206 A CN 201510277206A CN 105018428 A CN105018428 A CN 105018428A
- Authority
- CN
- China
- Prior art keywords
- cell
- cord blood
- mononuclearcell
- hematopoietic stem
- days
- Prior art date
- 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
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an in-vitro amplification method for cord blood hematopoietic stem cells. According to the invention, culture process is optimized and screened, so cultured cord blood hematopoietic stem cells can meet demands of populations needing a great number of cord blood hematopoietic stem cells, have longer life compared with hematopoietic stem cells obtained after bone marrow transplantation and have low incidence rate of graft-versus-host disease. The culturing method provided by the invention is simple and easily practicable and has low cost and good application effect.
Description
Technical field
The present invention relates to bio-science technical field, especially a kind of amplification in vitro method of umbilical cord blood hematopoietic stem cell.
Background technology
In recent years, the people attempt to replace grownup's marrow to transplant with mankind's umbilical cord blood hematopoietic stem cell (HSC), to treat some malignant diseases.But press marrow and minimum standard (1-2 × 10 needed for peripheral blood transplanting
8cell/kg body weight or 0.5-1.0 × 10
6cD34
+cell/body weight) calculate, the stem cell amount contained by every 100ml bleeding of the umbilicus can only meet the children being not more than 30kg, and limited and be restricted to adult's application factor amount.
Summary of the invention
The object of the invention is: the amplification in vitro method that a kind of umbilical cord blood hematopoietic stem cell is provided, HSCA after the method amplification, the incidence that crowd's needs that can not only meet large magnitude also have than having longer life-span and graft versus host disease (GVH disease) after bone marrow transplantation is simultaneously low.
The present invention is achieved in that the amplification in vitro method of umbilical cord blood hematopoietic stem cell, comprises the steps:
1) Cord blood and serum free medium are mixed by the volume ratio of 1:1; Be separated the mononuclearcell in Cord blood with the lymphocyte separation medium that proportion is 1.077, be separated lymphocyte separation medium with 50ml centrifuge tube and mix with Cord blood mixed solution, its volume ratio is 10:35, centrifugal 20 minutes of 2000rpm under room temperature;
2) draw mononuclearcell suspension, got mononuclearcell suspension 30ml physiological saline is washed twice, and centrifugal 10 minutes of each 1000rpm, abandons supernatant;
3) 1/3 amount of gained mononuclearcell is carried out Cell culture invitro, another 2/3 cell concentration carries out Liquid nitrogen storage;
4) Cell culture invitro serum free medium, and add stem cell factor 100ng/ml, thrombopoietin 100ng/ml and granulocyte stimulating factor 100ng/ml, cell concn is adjusted to 5x10
5/ ml; 37
oc, mass percent concentration is 5% CO
2cultivate 5 days in incubator;
5) mononuclearcell of frozen other 2/3 amount is recovered, join in the mononuclearcell of vitro culture and continue co-cultivation; Supply the concentration of stem cell factor, incubation time is 10 days simultaneously; In the middle of this 10 days cell cultivation process, every 3-4 days changes liquid once, supplies stem cell factor;
6) after cultivation terminates, cell physiological saline is washed 2 times, detect CD34+ cell count, and carry out Biosafety monitoring.
In order to verify technique effect of the present invention: the product that applicant adopts method according to the present invention to obtain is tested as follows:
Cord blood cells rebuilds mouse hemopoietic functional experiment
1, the foundation of mouse transplantation model
Mouse 24, in 8 one 10 week age, body weight 25 1 289, transplant and begin the last week, high pressure goes out
Adding gentamicin (400,000 units/L) and amphotericin B (50mg/L) mouse in bacterium tap water, to accept total dose be the full-body exposure of 2.75Gy Lethal Dose 50, and dose rate is 0.2Gy/min
Irradiation time is 1 hour, irradiates mouse and all after irradiation, feeds back transplanted cells in 4 hours,
Mouse survival is disconnected neck execution after 10 weeks, and wherein every 7 days extraction blood is done to detect and found that in blood, red corpuscle and leukocyte count grow steadily, and particularly CD34+ cell is also steady rise.
Then cord blood cells is through flow cytomery, and CD34+ positive percentage average is 92%, is up to 98%.The expression of purifying cells CD45 antigen is more weak, and CD34 antigen presentation is weak to medium, and cell volume is little, and intracellular granular is few, is one compared with the cell mass of integrated distribution.The cord blood CD34~+ cells of purifying is observed after Wright's staining, and cell size is homogeneous, and cell volume is less, and nuclear chromatin is dark, thick, and in strip and block, endochylema is little, and caryoplasm ratio is large, can not see particle in endochylema.
Owing to have employed above technical scheme, the present invention is optimized screening to culturing process, make its HSCA cultivating acquisition can not only meet crowd's needs of large magnitude, the incidence simultaneously also had than having longer life-span and graft versus host disease (GVH disease) after bone marrow transplantation is low.The present invention is simple, with low cost, and result of use is good.
Embodiment
Embodiments of the invention: the amplification in vitro method of umbilical cord blood hematopoietic stem cell, comprises the steps:
1) Cord blood and serum free medium are mixed by the volume ratio of 1:1; Be separated the mononuclearcell in Cord blood with the lymphocyte separation medium that proportion is 1.077, be separated lymphocyte separation medium with 50ml centrifuge tube and mix with Cord blood mixed solution, its volume ratio is 10:35, centrifugal 20 minutes of 2000rpm under room temperature;
2) draw mononuclearcell suspension, got mononuclearcell suspension 30ml physiological saline is washed twice, and centrifugal 10 minutes of each 1000rpm, abandons supernatant;
3) 1/3 amount of gained mononuclearcell is carried out Cell culture invitro, another 2/3 cell concentration carries out Liquid nitrogen storage;
4) Cell culture invitro serum free medium, and add stem cell factor 100ng/ml, thrombopoietin TPO100ng/ml and granulocyte stimulating factor 100ng/ml, cell concn is adjusted to 5x10
5/ ml; 37
oc, mass percent concentration is 5% CO
2cultivate 5 days in incubator;
5) mononuclearcell of frozen other 2/3 amount is recovered, join in the mononuclearcell of vitro culture and continue co-cultivation; Supply the concentration of stem cell factor, incubation time is 10 days simultaneously; In the middle of this 10 days cell cultivation process, every 3-4 days changes liquid once, supplies stem cell factor.
6) after cultivation terminates, cell physiological saline is washed 2 times, detect CD34+ cell count, and carry out Biosafety monitoring.
When the product obtained according to the present invention is defeated by patient, usual amounts is mononuclearcell number 10
7/ kg body weight, or CD34+ cell 105/kg body weight, adopt the slow drop infusion of disposable vein.
Of the present inventionly be not limited to the embodiment described in embodiment, those skilled in the art's technical scheme according to the present invention draws and other embodiment belongs to technological innovation scope of the present invention equally.Obvious those skilled in the art can carry out various change and modification to the present invention and not depart from the spirit and scope of the present invention.Like this, if these amendments of the present invention and modification belong within the scope of the claims in the present invention and equivalent technologies thereof, then the present invention is also intended to comprise these change and modification.
Claims (1)
1. an amplification in vitro method for umbilical cord blood hematopoietic stem cell, is characterized in that: comprise the steps:
1) Cord blood and serum free medium are mixed by the volume ratio of 1:1; Slowly add lymphocyte separation medium upper strata; Be separated the mononuclearcell in Cord blood with the lymphocyte separation medium that proportion is 1.077, in 50ml centrifuge tube, its volume ratio is 10:35, centrifugal 20 minutes of 2000rpm under room temperature;
2) draw mononuclearcell suspension, got mononuclearcell suspension 30ml physiological saline is washed twice, and centrifugal 10 minutes of each 1000rpm, abandons supernatant;
3) 1/3 amount of gained mononuclearcell is carried out Cell culture invitro, another 2/3 cell concentration carries out Liquid nitrogen storage;
4) Cell culture invitro serum free medium, and add stem cell factor 100ng/ml, thrombopoietin 100ng/ml and granulocyte stimulating factor 100ng/ml, cell concn is adjusted to 5x10
5/ ml; 37
oc, mass percent concentration is 5% CO
2cultivate 5 days in incubator;
5) mononuclearcell of frozen other 2/3 amount is recovered, join in the mononuclearcell of vitro culture and continue co-cultivation; Supply the concentration of stem cell factor, incubation time is 10 days simultaneously; In the middle of this 10 days cell cultivation process, every 3-4 days changes liquid once, supplies stem cell factor;
6) after cultivation terminates, cell physiological saline is washed 2 times, detect CD34+ cell count, and carry out Biosafety monitoring.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510277206.3A CN105018428A (en) | 2015-05-27 | 2015-05-27 | In-vitro amplification method for cord blood hematopoietic stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510277206.3A CN105018428A (en) | 2015-05-27 | 2015-05-27 | In-vitro amplification method for cord blood hematopoietic stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105018428A true CN105018428A (en) | 2015-11-04 |
Family
ID=54408733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510277206.3A Pending CN105018428A (en) | 2015-05-27 | 2015-05-27 | In-vitro amplification method for cord blood hematopoietic stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105018428A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106730015A (en) * | 2017-01-06 | 2017-05-31 | 朱凯林 | A kind of preparation for beauty and preparation method thereof |
| 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 |
| CN110343663A (en) * | 2019-07-29 | 2019-10-18 | 山东省齐鲁干细胞工程有限公司 | A method of total karyocyte and mononuclearcell are separated from Cord blood |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1280187A (en) * | 1999-07-13 | 2001-01-17 | 中国人民解放军第二军医大学 | Method of extracorporeal cloning hemopoietic stem cell |
| WO2004046312A2 (en) * | 2002-11-15 | 2004-06-03 | The Board Of Trustees Of The University Of Illinois | Methods for in vitro expansion of hematopoietic stem cells |
| CN1556197A (en) * | 2003-12-30 | 2004-12-22 | 上海伯瑞生物技术发展有限公司 | Method of prodcing megacaryocyle using umbilical blood CD 344+cell in vitro induction method |
| US20130136722A1 (en) * | 2011-11-11 | 2013-05-30 | The Board Of Trustees Of The University Of Illinois | Methods of Ex Vivo Expansion of Blood Progenitor Cells, and Generation of Composite Grafts |
| WO2014123930A2 (en) * | 2013-02-05 | 2014-08-14 | Williams Joseph L | Prevention and treatment of tissue fibrosis |
-
2015
- 2015-05-27 CN CN201510277206.3A patent/CN105018428A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1280187A (en) * | 1999-07-13 | 2001-01-17 | 中国人民解放军第二军医大学 | Method of extracorporeal cloning hemopoietic stem cell |
| WO2004046312A2 (en) * | 2002-11-15 | 2004-06-03 | The Board Of Trustees Of The University Of Illinois | Methods for in vitro expansion of hematopoietic stem cells |
| CN1556197A (en) * | 2003-12-30 | 2004-12-22 | 上海伯瑞生物技术发展有限公司 | Method of prodcing megacaryocyle using umbilical blood CD 344+cell in vitro induction method |
| US20130136722A1 (en) * | 2011-11-11 | 2013-05-30 | The Board Of Trustees Of The University Of Illinois | Methods of Ex Vivo Expansion of Blood Progenitor Cells, and Generation of Composite Grafts |
| WO2014123930A2 (en) * | 2013-02-05 | 2014-08-14 | Williams Joseph L | Prevention and treatment of tissue fibrosis |
Non-Patent Citations (1)
| Title |
|---|
| CHAOLING YAO ET AL.: "Factorial designs combined with the steepest ascent method to optimize serum-free media for ex vivo expansion of human hematopoietic progenitor cells", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106730015A (en) * | 2017-01-06 | 2017-05-31 | 朱凯林 | A kind of preparation for beauty and preparation method thereof |
| 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 |
| CN110343663A (en) * | 2019-07-29 | 2019-10-18 | 山东省齐鲁干细胞工程有限公司 | A method of total karyocyte and mononuclearcell are separated from Cord blood |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103756963B (en) | A kind of method of amplification in vitro NK cell | |
| Hanley et al. | Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System | |
| CN108220239B (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN108893443A (en) | A kind of Efficient amplification method of cytokine induction umbilical cord blood natural killer | |
| CN101519646B (en) | CIK cell, as well as preparation method and cell preparation thereof | |
| CN104371972B (en) | A kind of non-animal derived property and humanized's composition T lymphocytes culture medium and preparation method thereof | |
| CN102827810B (en) | Non-animal-source serum-free culture medium for umbilical cord blood stem cells | |
| CN107267454A (en) | The amplification in vitro method and its kit of a kind of Cord Blood Natural Killer Cells: Impact and application | |
| CN104357390A (en) | Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells | |
| CN102539736B (en) | CD106-positive cells, and identification and preparation method and application thereof | |
| CN102268405A (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| WO2020027163A1 (en) | Method for producing dental pulp-derived cells | |
| CN107502590A (en) | A kind of method of human umbilical cord's blood candidate stem cell efficient amplification NK cells | |
| KR20170139095A (en) | Method for manufacturing immunocyte-containing composition, and cancer-treating composition | |
| CN105543169A (en) | Preparation method and application of human TSCMs (T memory stem cells) | |
| CN104894072B (en) | A kind of preparation method and applications of autologous natural killer cells propagation | |
| CN105018428A (en) | In-vitro amplification method for cord blood hematopoietic stem cells | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation | |
| EP2878666A1 (en) | Industrial preparation of natural killer cells (nks) and injection using human allogeneic karyocytes | |
| CN102154205A (en) | Method for preparing high-purity and high-cytotoxin-activity gamma delta T cells | |
| CN110272871A (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN103981144A (en) | Preparation method for autologous-serum antigen-sensitized DC-CIK cells | |
| CN102154208A (en) | Preparation method and use of cord blood-derived (CD)133 and brain glioma stem cell antigen carrying dendritic cells | |
| CN104762261A (en) | Tumor infiltrating lymphocytes separation method | |
| KR101202836B1 (en) | Method for expansion of hematopoietic stem cells and progenitor cells using blood mononuclear cell sphere and stem and progenitor cells produced by the method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 550005 No. 109 Duyun Road, Guiyang High-tech Zone, Guizhou Province Applicant after: Guizhou broad cell Cell Biotechnology Co., Ltd. Address before: 550005 No. 109 Duyun Road, Guiyang High-tech Zone, Guizhou Province Applicant before: Ke Fanteer bio tech ltd, north, Guizhou |
|
| CB02 | Change of applicant information | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151104 |
|
| RJ01 | Rejection of invention patent application after publication |