CN102363764A - A method for culturing pancreatic stem/progenitor cells - Google Patents
A method for culturing pancreatic stem/progenitor cells Download PDFInfo
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- CN102363764A CN102363764A CN2011103445554A CN201110344555A CN102363764A CN 102363764 A CN102363764 A CN 102363764A CN 2011103445554 A CN2011103445554 A CN 2011103445554A CN 201110344555 A CN201110344555 A CN 201110344555A CN 102363764 A CN102363764 A CN 102363764A
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Abstract
本发明公开了一种培养胰腺干/祖细胞的方法，包括以下步骤：将胰腺干/祖细胞接种于微孔培养板中，接种结束后，立即快速将接种板翻转，使接种的细胞悬液和培养液的混合物在接种板表面形成吸附于接种板的向下的悬滴，而不至于导致液体滴落或溅出；在悬滴状态下培养至细胞形成细胞球之后，将细胞球转移至漂浮的培养膜上继续培养至形成具有生理学功能的结构。本发明的悬滴培养法简单方便，不需要特定的生物材料，而且细胞间的接触更为紧密，能够更好地模拟体内生长环境。
The invention discloses a method for cultivating pancreatic stem/progenitor cells, which comprises the following steps: inoculating pancreatic stem/progenitor cells in a microporous culture plate, and immediately turning over the inoculated plate rapidly after the inoculation, so that the inoculated cell suspension The mixture with the culture medium forms a downward hanging drop adsorbed on the seeding plate on the surface of the seeding plate, so as not to cause the liquid to drip or splash; after the cells are cultured in the hanging drop state until the cells form a cell ball, transfer the cell ball to Continue culturing on the floating culture membrane until a structure with physiological functions is formed. The hanging drop culture method of the present invention is simple and convenient, does not require specific biological materials, and the contact between cells is closer, which can better simulate the growth environment in vivo.
Description
Technical field
The present invention relates to a kind of cultural method of cell, especially a kind of efficient induction pancreatic stem/progenitor cells is divided into the cultural method of endocrine cell.
Background technology
It is of paramount importance experimental technique in the biology that histocyte is cultivated, and uses very extensively, is promoting the development of numerous front line sciences such as molecular biology, genetically engineered, protein engineering etc.The histocyte best cultivation mainly is divided three classes: hanging drop cultivation, monolayer culture and stereoscopic culture.
The hanging drop culture method: 20 beginnings of the century were founded single cover plate hanging drop culture method by Harrison and Carrel, and nineteen twenty-four Maximow improvement is double-canopy hanging drop culture method.The advantage that hanging drop is cultivated is that cell is grown in and is similar in the spatial environment, and defective is that the space of cell growth is narrow and small, gas is not enough and nutritive ingredient is few, the growth of restrictive cell.
Monolayer method: since phase late 1940s,, use the artificial synthetic medium instead and add foetal calf serum and in culturing bottle, cultivate, can make cell be grown to individual layer, and become monolayer culture as nutrient solution with cell inoculation by the scholars headed by the Dulbecco.Monolayer culture has and goes down to posterity conveniently, is easy to variety of methods and observe and study, so that become the world's classical cultural method of widespread usage still so far.The cell of monolayer culture has only long and wide two-dirnentional structure, has lost the solid shape when substance is interior, causes cell can not give full expression to the corresponding gene product and breaks up.
The stereoscopic culture method: at present, stereoscopic culture is still in improving and developing.The advantage of this method is: with cell cultures in special container, can updated at any time nutrient solution, nutritive substance is provided and gets rid of meta-bolites; For culturing cell provide with body in similar mounting system, build up with substance in similar living environment; For culturing cell provides specific short cell proliferation growth and differentiation factor.
2007, the sickness rate of China's mellitus increased to 5%, and the mortality ratio of mellitus and complication thereof has risen to the 3rd.Along with the development of pancreatic islets transplantation technology, pancreatic islets transplantation becomes thorough treatment mellitus method the most likely.But the deficiency of donor pancreas has limited extensively carrying out of pancreatic islets transplantation, therefore, presses for the new source of seeking the β cell.Pancreatic stem/progenitor cells can be divided into the β cell, so the separation of pancreatic stem/progenitor cells, cultivates and induce differentiation to provide the foundation for the cell therapy mellitus.At present, pancreatic stem/progenitor cells still lacks special surface marker, and the investigator is attempting seeking.Use special surface marker, flow cytometer or immunological magnetic bead sorting can be simple, separate pancreatic stem/progenitor cells effectively.But; The greatest difficulty that faces behind the separation pancreatic stem/progenitor cells is that the cell count that obtains is fewer; Most of investigator adopts conventional cultural method such as monolayer culture or dimensional culture, and the efficient of inducing it to be divided into the β cell is low, and needs inducing of a large amount of cytokines.
Summary of the invention
The efficient that exists in the existing pancreatic stem/progenitor cells culture technique is low, cytokine induction dosage big in order to solve, the defectives such as the few hypofunction of β cell quantity after inducing; The invention provides a solution; Hanging drop culture method and stereoscopic culture method are combined and improve, set up a kind of method of cultivating pancreatic stem/progenitor cells.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of method of cultivating pancreatic stem/progenitor cells may further comprise the steps:
(1) pancreatic stem/progenitor cells is inoculated in the well plates; After inoculation finishes; To inoculate plate upset immediately fast, and make the cell suspension of inoculation and the mixture of nutrient solution form the downward hanging drop that is adsorbed in the inoculation plate, and be unlikely to cause liquid to drip or spill on inoculation plate surface;
(2) under the hanging drop state, be cultured to cell and form after the cell ball, the cell ball is transferred to continue on the buoyant culture membrane to be cultured to form has the structure of physiologic function.
Specifically, may further comprise the steps:
(1) dissecting microscope separates the pancreas of pregnant 12.5-17.5 days fetal mice down; Adding final concentration is the collagenase IV 200 μ l of 250U/ml; Hatch 30min for 37 ℃, add the PRMI-1640 perfect medium and stop digestion, blow and beat embryonic pancreas repeatedly until no obvious sediment with the 1ml syringe; Behind the 100 purpose strainer filterings, the single cell suspension of preparation pancreatic cell;
(2) concentration of adjustment cell suspension is (1-5) * 10
3It is 0.013cm that/μ l, obtained cell suspension are inoculated in floorage
2Well plates in, after inoculation finishes, reverse culture plate fast in 20-30 μ l/ hole, iuntercellular each other closely contact form a cell ball;
(3) after inverted well plates is cultivated 18-24h, be transferred on the LCR film that the buoyant diameter is 25mm at following cell ball of dissecting microscope, diameter is an adding 2mlPRMI-1640 perfect medium in the little petridish of 35mm; The LCR film floats in the substratum; Cell spheroid was cultivated six days on buoyant LCR film, and the embryonic pancreas stem cell is divided into inside and outside secretory cell gradually, contains a large amount of secretory granules in the cell; Cultivate after 7 days, carry out immunohistochemistry and detect ripe pancreatic cell.
Add 10% foetal calf serum by volume in the said PRMI-1640 perfect medium, 100U/ml penicillium mould, 100ug/ml Streptomycin sulphate, the Stimulina of 1% non-essential amino acid and 2mM.
The invention has the beneficial effects as follows: the hanging drop technique among the present invention; Not only be fit to the cultivation of small amounts of cells; Can efficiently be divided into endocrine cell at external evoked pancreatic stem cells; And do not need inducible factor, can make the contact between the pancreatic cell tightr, for the growth of pancreatic cell, propagation provide good three-dimensional environment.The dimensional culture technology that the contrast organization material provides, the hanging drop culture method is simple and convenient, does not need specific biomaterial, and intercellular contact is more tight, better growing environment in the analogue body.Owing to possessed the similarity of biological characteristics and growth pattern between the different sorts stem cell,, also be applicable to the cultivation of other kind stem cell so this cultural method not only can be used for the cultivation of pancreatic cell.
Description of drawings
The synoptic diagram that Fig. 1 hanging drop is cultivated;
Fig. 2 a cell just has been inoculated in the photo of culture plate;
The photo of Fig. 2 b cell inoculation after in hanging drop, cultivating 1 day behind the culture plate;
The synoptic diagram that Fig. 3 cell ball is cultivated on the buoyant film;
Fig. 4 cell ball is the variation of form in the culturing process on the buoyant film;
Fig. 5 cell ball pancreatic cell sign change of Expression in the culturing process on the buoyant film.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further explain:
Embodiment of the present invention is following, but enforcement of the present invention is not limited to following examples:
Dissecting microscope separates the pancreas of fetal mice (pregnant 12.5-17.5 days) down, and adding final concentration is the collagenase IV 200 μ l of 250U/ml, hatches 30min for 37 ℃; Add the PRMI-1640 perfect medium and (add 10% foetal calf serum, 100U/ml penicillium mould, 100ug/ml Streptomycin sulphate; The Stimulina of 1% non-essential amino acid and 2mM) stops digestion; Blow and beat embryonic pancreas repeatedly until no obvious sediment with the 1ml syringe, behind the 100 purpose strainer filterings, the single cell suspension of preparation pancreatic cell.The concentration of adjustment cell suspension is 1-5 * 10
3/ μ l, obtained cell suspension are inoculated in (Nunclon Microwell Terasaki-Style Plates) in the well plates that floorage is 0.013cm2,20-30 μ l/ hole; After inoculation finishes; Reverse culture plate fast, because capillary effect, nutrient solution can not flow down.Because action of gravity, iuntercellular closely contact each other and form a cell ball (Fig. 1,2).After inverted well plates is cultivated 18-24h, be transferred on the LCR film that the buoyant diameter is 25mm at following cell ball of dissecting microscope.Diameter is to add 2ml PRMI-1640 perfect medium in the little petridish of 35mm, and the LCR film floats on (Fig. 3) in the substratum.Cell spheroid was cultivated six days on buoyant LCR film, the variation of cell ball such as Fig. 4 in the culturing process.Although the volume change of cell spheroid is little in culturing process, along with the increase of cultivating fate, its density increases gradually.Because along with the increase of cultivating fate, the embryonic pancreas stem cell is divided into inside and outside secretory cell gradually, contains a large amount of secretory granules in the cell.Cultivate after 7 days, immunohistochemistry detects the sign of ripe pancreatic cell and finds cell ball high expression level Regular Insulin, hyperglycemic-glycogenolytic factor and carboxypeptidase, and the flock together structure of the similar pancreas islet of formation of Regular Insulin positive cell and hyperglycemic-glycogenolytic factor positive cell.The expression of cell ball culturing process Regular Insulin raises gradually, and first decline of the expression of hyperglycemic-glycogenolytic factor afterwards raises.Because hyperglycemic-glycogenolytic factor is considered to be expressed in pancreatic stem cells.Along with pancreatic stem cells differentiation hyperglycemic-glycogenolytic factor is expressed decline gradually, express minimum in the time of the 3rd day.But along with ripe α cell occurs, hyperglycemic-glycogenolytic factor is expressed gradually to be increased.In the cell ball culturing process in the expression pattern of Regular Insulin and hyperglycemic-glycogenolytic factor and the body in the embryonic pancreas growth course consistent (Fig. 5).
In sum, content of the present invention is not confined in the above embodiments, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment is included within the scope of the present invention.
Claims (3)
1. a method of cultivating pancreatic stem/progenitor cells is characterized in that, may further comprise the steps:
(1) pancreatic stem/progenitor cells is inoculated in the well plates; After inoculation finishes; To inoculate plate upset immediately fast, and make the cell suspension of inoculation and the mixture of nutrient solution form the downward hanging drop that is adsorbed in the inoculation plate, and be unlikely to cause liquid to drip or spill on inoculation plate surface;
(2) under the hanging drop state, be cultured to cell and form after the cell ball, the cell ball is transferred to continue on the buoyant culture membrane to be cultured to form has the structure of physiologic function.
2. the method for cultivation pancreatic stem/progenitor cells according to claim 1 is characterized in that, may further comprise the steps:
(1) dissecting microscope separates the pancreas of pregnant 12.5-17.5 days fetal mice down; Adding final concentration is the collagenase IV 200 μ l of 250U/ml; Hatch 30min for 37 ℃, add the PRMI-1640 perfect medium and stop digestion, blow and beat embryonic pancreas repeatedly until no obvious sediment with the 1ml syringe; Behind the 100 purpose strainer filterings, the single cell suspension of preparation pancreatic cell;
(2) concentration of adjustment cell suspension is (1-5) * 10
3It is 0.013cm that/μ l, obtained cell suspension are inoculated in floorage
2Well plates in, after inoculation finishes, reverse culture plate fast in 20-30 μ l/ hole, iuntercellular each other closely contact form a cell ball;
(3) after inverted well plates is cultivated 18-24h, be transferred on the LCR film that the buoyant diameter is 25mm at following cell ball of dissecting microscope, diameter is an adding 2mlPRMI-1640 perfect medium in the little petridish of 35mm; The LCR film floats in the substratum; Cell spheroid was cultivated six days on buoyant LCR film, and the embryonic pancreas stem cell is divided into inside and outside secretory cell gradually, contains a large amount of secretory granules in the cell; Cultivate after 7 days, carry out immunohistochemistry and detect ripe pancreatic cell.
3. the method for cultivation pancreatic stem/progenitor cells according to claim 1; It is characterized in that, add 10% foetal calf serum by volume in the said PRMI-1640 perfect medium, 100U/ml penicillium mould; The 100ug/ml Streptomycin sulphate, the Stimulina of 1% non-essential amino acid and 2mM.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106029862A (en) * | 2014-02-25 | 2016-10-12 | 株式会社可乐丽 | Spheroid manufacturing device, method of recovering spheroid, and manufacturing method |
| CN108486035A (en) * | 2018-03-26 | 2018-09-04 | 西北大学 | A kind of drop cultural method of three-dimensional organoid |
| CN110257318A (en) * | 2019-05-27 | 2019-09-20 | 上海长海医院 | A kind of preparation method of chronic pancreatitis mice pancreatic single cell suspension |
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2011
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106029862A (en) * | 2014-02-25 | 2016-10-12 | 株式会社可乐丽 | Spheroid manufacturing device, method of recovering spheroid, and manufacturing method |
| CN108486035A (en) * | 2018-03-26 | 2018-09-04 | 西北大学 | A kind of drop cultural method of three-dimensional organoid |
| CN108486035B (en) * | 2018-03-26 | 2021-11-26 | 西北大学 | Liquid drop culture method of three-dimensional organoid |
| CN110257318A (en) * | 2019-05-27 | 2019-09-20 | 上海长海医院 | A kind of preparation method of chronic pancreatitis mice pancreatic single cell suspension |
| CN110257318B (en) * | 2019-05-27 | 2023-05-30 | 上海长海医院 | A preparation method of pancreas single cell suspension in mice with chronic pancreatitis |
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Application publication date: 20120229 |