CN105002138A - Three-dimensional cell culture method for megakaryocyte progenitor induced in vitro - Google Patents
Three-dimensional cell culture method for megakaryocyte progenitor induced in vitro Download PDFInfo
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- CN105002138A CN105002138A CN201510446748.9A CN201510446748A CN105002138A CN 105002138 A CN105002138 A CN 105002138A CN 201510446748 A CN201510446748 A CN 201510446748A CN 105002138 A CN105002138 A CN 105002138A
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Abstract
The invention discloses a three-dimensional cell culture method for megakaryocyte progenitor induced in vitro. The method includes the steps of cultivating mononuclear cells in a cell culture bag, and placing the cell culture bag on a seesaw type biological reactor for culture, wherein the oscillation angle of the seesaw type biological reactor ranges from 2 degrees to 13 degrees, and the rotating speed of the seesaw type biological reactor ranges from 3 rpm to 40 rpm. The mononuclear cells induced through the three-dimensional cell culture method are converted into megakaryocyte progenitor, and the induction efficiency is remarkably improved.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to the method for cell cultures, more specifically, relate to the Three-dimensional cell culture method of external evoked megakaryoblast.
Background technology
Thrombocyte is cell minimum in blood, have and prevent hemorrhage and reduce the vital role of blood vessel injury, tumor incidence is in rising trend in recent years, often there is thrombopenia in the inspection of tumor chemoradiotherapy patient blood, clinically often through infusion Single-donor platelets as treatment means, because blood source is nervous and need platelet transfusion repeatedly, us are forced to find new thrombocyte source.Hemopoietic stem cell is the hemopoietic forebody cell with self and multi-lineage potential, at present for treatment that the is pernicious and various diseases such as non-malignant hematological disorder, noumenal tumour.Hemopoietic stem cell can derive from marrow, peripheral blood and Cord blood etc., and wherein umbilical cord blood hematopoietic stem cell has that multiplication capacity is strong, immunogenicity is low, be easy to the advantages such as acquisition, has become the focus that people pay close attention to.Umbilical hemopoietic stem cell can be divided into megakaryoblast, and generation thrombocyte of reaching maturity further, be therefore expected to by external feedback megakaryoblast to improve hematoblastic quantity in blood, alleviate or the nervous aleukia caused in Blood substitute source.
Therefore, the method for external evoked Megakaryocyte awaits further research.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is a kind of Three-dimensional cell culture method proposing external evoked megakaryoblast.
It should be noted that, the present invention completes based on the following work of contriver:
Contriver finds, the Cord Blood Mononuclear Cell of separation is inoculated in two kinds of cell culture bags in WIGGENS shaking table and WAVE bio-reactor, carries out three-dimensional concussion cultivation respectively, cultivate as contrast with static cell bottle two dimension, observation two dimension is cultivated and is induced megakaryoblast with dimensional culture simultaneously.Research finds, adopts dimensional culture mode to induce megakaryoblast, can significantly improve induced efficiency
Thus, according to an aspect of the present invention, the invention provides a kind of Three-dimensional cell culture method of external evoked megakaryoblast.According to embodiments of the invention, the method comprises and being incubated in cell culture bags by mononuclearcell, and is placed on seesaw type bio-reactor and cultivates, and wherein, the oscillation angle of described seesaw type bio-reactor is 2-13 °, and rotating speed is 3-40rpm.
The discovery that contriver is surprised, adopt this Three-dimensional cell culture method to induce mononuclearcell to be converted into megakaryoblast, induced efficiency significantly improves.
According to embodiments of the invention, described oscillation angle is 5 °.
According to embodiments of the invention, described rotating speed increases with the prolongation of cell cultures time.Thus, the propagation of cell, Induction and differentiation is more suitable for.
According to embodiments of the invention, at the 1-10 days of cell cultures, described rotating speed is 10rpm; After the 10th day of cell cultures, described rotating speed is 14rpm.Thus, cell proliferation rate is fast.
According to embodiments of the invention, for cultivating the substratum of mononuclearcell be: containing the stemspan serum free medium of SCF, IL-3, IL-6, TPO.Thus, culture environment is suitable for Growth of Cells.
According to embodiments of the invention, the concentration of described SCF is 20 ~ 200ng/mL, preferred 50ng/mL; The concentration of described IL-3 is 5 ~ 100ng/mL, preferred 20ng/mL; The concentration of described IL-6 is 10 ~ 200ng/mL, preferred 50ng/mL; The concentration of described rhTPO is 20 ~ 200ng/mL, preferred 50ng/mL.Thus, culture environment is more suitable for Growth of Cells.
According to embodiments of the invention, the described substratum for cultivating mononuclearcell, comprises: PLURONICS F87 further.Thus, to be divided into the efficiency of megakaryoblast higher for cell induction.
According to embodiments of the invention, the concentration of described PLURONICS F87 (poloxamer 188, P188) is 0.2 ~ 2mg/mL, preferred 1mg/mL.Thus, high cell growth speed, the efficiency that cell induction is divided into megakaryoblast significantly improves
According to embodiments of the invention, respectively at the 4th day, the 7th day, the 10th day of cell cultures, in described cell culture bags, supplement the substratum of 1/3 volume of former substratum.Thus, cell proliferation rate is fast.
According to embodiments of the invention, the kind of described seesaw type bio-reactor is not particularly limited, as long as angle can be provided to be 2-13 DEG C for cell culture bags, rotating speed is the vibration of 3-40rpm, such as: seesaw shaking table and WAVE bio-reactor.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the expression of cell surface marker CD41, CD61 of the external 2D training method induction of flow cytomery according to an embodiment of the invention megakaryoblast;
Fig. 2 shows the expression of cell surface marker CD41, CD61 of the external 3D training method induction of flow cytomery according to an embodiment of the invention megakaryoblast;
Fig. 3 shows the expression of cell surface marker CD41, CD61 of the external 3D+P188 training method induction of flow cytomery according to an embodiment of the invention megakaryoblast.
Fig. 4 shows the cellular form of 2D inducing culture 14d Switzerland Ji nurse Sa dyeing according to an embodiment of the invention.
Fig. 5 shows 3D inducing culture 14d according to an embodiment of the invention, the cellular form of Switzerland Ji nurse Sa dyeing.
Embodiment
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments are only illustrative, and can not be interpreted as limitation of the present invention.
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1
One, material
Cord blood: 70ml originates Beijing Cord Blood Bank, acquisition units Tongzhou healthcare hospital for women & children;
Permeability type culture bag: article No. KB0005,300ml, Bao Yi Bioisystech Co., Ltd, Japan produces;
WIGGENS shaking table: seesaw vibrator, WS-350 Germany produces;
CO
2cell culture incubator: Thermo Scientific Model 3111.
Two, method
Be separated human umbilical cord blood mononuclear cell, then, adopt cell bottle (2D), cell culture bags (3D), cell culture bags (3D+P188) three kinds of methods to cultivate it respectively, concrete grammar is as follows:
1, cell bottle (2D) is cultivated:
1) substratum: containing the Stemspan substratum of 50ng/mL SCF, 20ng/mL IL-3,50ng/mL IL-6,50ng/mL TPO
2) cultural method:
Mononuclearcell is cultivated in culturing bottle, culture volume 20ml, cell density 2x10
6/ ml, then, by cell bottle static gas wave refrigerator, and in 4d, 7d, 10d by 1/3 fluid infusion.
2, cell culture bags (3D) is cultivated:
1) substratum: containing the Stemspan substratum of 50ng/mL SCF, 20ng/mL IL-3,50ng/mL IL-6,50ng/mL TPO,
2) cultural method:
Mononuclearcell is cultivated in culture bag, culture volume 20ml, cell density 2x10
6/ ml, then, cultivates cell culture bags on seesaw shaking table, angle 5 ° (2-13 °, preferred 5-7 °), rotating speed 10rpm, and in 4d, 7d, 10d by 1/3 fluid infusion, after 10d, rotating speed is increased to 14rpm.
3, cell culture bags (3D+P188) is cultivated:
1) substratum: containing the stemspan substratum of 1mg/mL P188,50ng/mL SCF, 20ng/mL IL-3,50ng/mL IL-6,50ng/mL TPO,
2) cultural method: identical with above-mentioned cell culture bags (3D) cultural method.
According to above-mentioned three kinds of methods, external evoked megakaryoblast 14d, then collecting cell, detects cell survival rate, cell count, cell surface marker.
Three, result
1, cellular form
Adopt the form of inverted microscope observation of cell, wherein, in 2D cultured cells bottle, mononuclearcell cultivates 4d, have attached cell, 7d cell count increases, and cell becomes large, attached cell is increased, and 10d packed cell increases, and cultivates 14d, dye to cell with Switzerland's Jim Sa, under microscope, the form of cell as shown in Figure 4, most cells volume is little, core similar round, and nuclear chromatin loosens, granule type cell is rare, and megakaryoblast quantity is few.Cell culture bags (3D) cultivate and cell culture bags (3D+P188) cultured cells culture bag in, cellular form and growth conditions are comparatively consistent, cultivate the agglomerating growth of 4d visible cell, have a small amount of red colony, 7d cell is agglomerating, and cell is larger; Cultivate 14d, dye with Switzerland's Jim Sa to cell, under microscope, as shown in Figure 5, nuclear-cytoplasmic ratio increases the form of cell, and cell volume becomes large, and nuclear shapes is irregular, occurs the cell containing particle, and megakaryoblast quantity increases.
2. cytobiology detects
After cell cultures 14d, to the cell surface marker CD41 of the megakaryoblast that above-mentioned three kinds of methods induction obtains, the expression of CD61 detects, the results are shown in Table 1-3 and Fig. 1-3, result shows, compared with cultivating with 2D, the megakaryoblast that 3D cultivates (comprising cell culture bags (3D) to cultivate and cell culture bags (3D+P188) cultivation) induces, cell surface marker CD41, CD61, the expression of CD41/CD61 is significantly increased, and it is more obvious to add P188 (namely cell culture bags (3D+P188) is cultivated) expression effect, and add P188 cell culture bags cultured cells survival rate and cell count and cell bottle and cultivate indifference.
The external evoked cell survival rate of table 1 and cell count detect
The external evoked cell surface marker of table 2 detects
*difference highly significant compared with cultivating with 2D.
＄ ＄difference highly significant compared with cultivating with 3D.
Table 3 different cell cultures pattern is on the impact of external evoked human cord blood megakaryoblast phenotype
*differ remarkable compared with cultivating with 2D.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. the Three-dimensional cell culture method of an external evoked megakaryoblast, it is characterized in that, mononuclearcell is incubated in cell culture bags, and be placed on seesaw type bio-reactor and cultivate, wherein, the oscillation angle of described seesaw type bio-reactor is 2-13 degree, and rotating speed is 3-40rpm.
2. Three-dimensional cell culture method according to claim 1, is characterized in that, described oscillation angle is 5 degree.
3. Three-dimensional cell culture method according to claim 1, is characterized in that, described rotating speed increases with the prolongation of cell cultures time.
4. Three-dimensional cell culture method according to claim 3, is characterized in that,
At the 1-10 days of cell cultures, described rotating speed is 10rpm;
After the 10th day of cell cultures, described rotating speed is 14rpm.
5. Three-dimensional cell culture method according to claim 1, is characterized in that, is: containing the stemspan serum free medium of SCF, IL-3, IL-6 and TPO for cultivating the substratum of mononuclearcell.
6. Three-dimensional cell culture method according to claim 5, is characterized in that,
The concentration of described SCF is 20 ~ 200ng/mL, preferred 50ng/mL;
The concentration of described IL-3 is 5 ~ 100ng/mL, preferred 20ng/mL;
The concentration of described IL-6 is 10 ~ 200ng/mL, preferred 50ng/mL;
The concentration of described TPO is 20 ~ 200ng/mL, preferred 50ng/mL.
7. the Three-dimensional cell culture method according to claim 5 or 6, is characterized in that, the described substratum for cultivating mononuclearcell, comprises: PLURONICS F87 further.
8. Three-dimensional cell culture method according to claim 7, is characterized in that, the concentration of described PLURONICS F87 is 0.2 ~ 2mg/mL, preferred 1mg/mL.
9. Three-dimensional cell culture method according to claim 1, is characterized in that, respectively at the 4th day, the 7th day, the 10th day of cell cultures, supplements the substratum of 1/3 volume of former substratum in described cell culture bags.
10. Three-dimensional cell culture method according to claim 1, is characterized in that, described seesaw type bio-reactor is seesaw shaking table or WAVE bio-reactor.
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| CN109628370A (en) * | 2018-12-30 | 2019-04-16 | 深圳光彩生命工程技术有限公司 | A kind of Three-dimensional cell culture method |
| CN113046310A (en) * | 2020-12-30 | 2021-06-29 | 中国人民解放军军事科学院军事医学研究院 | Culture medium and method for promoting hematopoietic stem cells to be induced and differentiated into megakaryocyte by shaking culture |
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| CN104232582A (en) * | 2014-08-27 | 2014-12-24 | 军事医学科学院华南干细胞与再生医学研究中心 | Application of poloxamer in inducing haemopoietic stem progenitor cell proliferation and/or meganucleus differentiation |
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| CN104232582A (en) * | 2014-08-27 | 2014-12-24 | 军事医学科学院华南干细胞与再生医学研究中心 | Application of poloxamer in inducing haemopoietic stem progenitor cell proliferation and/or meganucleus differentiation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628370A (en) * | 2018-12-30 | 2019-04-16 | 深圳光彩生命工程技术有限公司 | A kind of Three-dimensional cell culture method |
| CN113046310A (en) * | 2020-12-30 | 2021-06-29 | 中国人民解放军军事科学院军事医学研究院 | Culture medium and method for promoting hematopoietic stem cells to be induced and differentiated into megakaryocyte by shaking culture |
| CN113046310B (en) * | 2020-12-30 | 2023-08-18 | 中国人民解放军军事科学院军事医学研究院 | Culture medium and method for promoting hematopoietic stem cell induction differentiation into megakaryocyte by shaking culture |
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Application publication date: 20151028 |