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CN-121406569-B - Induction medium and method for rapidly obtaining spontaneously beating myocardial balls

CN121406569BCN 121406569 BCN121406569 BCN 121406569BCN-121406569-B

Abstract

The invention relates to the technical field of cell differentiation and the technical field of myocardial tissue regeneration medicine, in particular to an induction medium and a method for rapidly obtaining spontaneous beating myocardial balls, which are characterized in that myocardial cells from hiPSC sources are obtained by co-culture with hMSC, and the myocardial cells harvested by D7 are cloned more independently and are separated from other non-myocardial cells naturally, so that myocardial clones can be rapidly screened and purified by using physical and enzyme-free reagents, the step of screening and purifying the culture medium of the traditional method is omitted, and the time and the differentiation cost are saved.

Inventors

  • YANG YIXING
  • ZHANG HAN
  • HOU JUAN
  • CHEN XU
  • CHEN GANG

Assignees

  • 苏州依科赛生物科技股份有限公司
  • 上海吉泰依科赛生物科技有限公司

Dates

Publication Date
20260508
Application Date
20251230

Claims (8)

  1. 1. An induction culture medium capable of rapidly obtaining spontaneous beating myocardial balls is characterized by comprising an MSC expansion culture medium MH01, a pluripotent stem cell expansion culture medium mTESR1, a 10 mu M Y27632, a myocardial cell maintenance culture medium, a basal culture medium, a differentiation culture medium A, a differentiation culture medium B, a differentiation culture medium C and a differentiation culture medium D; Wherein, the Basic culture medium, DMEM/F12 culture medium +50 μg/mL vitamin C +1XB27-insulin +100deg.X trace element B; Differentiation medium A basal medium+6mu.M Chir99021; differentiation Medium B basal Medium+2.5. Mu.M IWR1+2.5. Mu.M IWP2+4mM HCl; Differentiation medium C basal medium+4 mM hydrochloric acid; The differentiation medium D is DMEM/F12 medium+50 mug/mL vitamin C+1XB27+100Xtrace element B; Cardiomyocyte maintenance medium RPMI 1640 medium +1XB27+50. Mu.g/mL vitamin C +5ng/mL FGF2.
  2. 2. An induction culture method for rapidly obtaining spontaneously beating myocardial balls, characterized in that the induction culture is performed using an induction culture medium for rapidly obtaining spontaneously beating myocardial balls as defined in claim 1.
  3. 3. An induction culture method for rapidly obtaining spontaneously beating myocardial balls according to claim 2, comprising the following stages: the first stage was to co-culture hiPSC and hMSC in 2D culture to obtain spontaneously beating cardiomyocytes.
  4. 4. A method of induced culture for rapidly obtaining spontaneously beating myocardial balls according to claim 3, wherein the ratio of the initial cell amount of hiPSC to the initial cell amount of hMSC is 5:3.
  5. 5. The method for inducing and culturing myocardial balls capable of rapidly acquiring spontaneous beating according to claim 4, wherein the initial cell amount of hiPSC is 5 ten thousand per square centimeter and the initial cell amount of hMSC is 3 ten thousand per square centimeter.
  6. 6. A method of inducing and culturing a rapidly beating myocardial ball according to claim 3 wherein the first stage comprises the steps of: 1) In the MSC plating stage, a stem cell matrigel coated cell culture plate is prepared in advance, hMSC from P3-P5 generation human umbilical cord separation sources is selected and planted in a coated pore plate, and MSC expansion culture medium MH01 is added for overnight culture; 2) The hPSC single-layer inoculation comprises the steps of selecting hiPSC with good state, detecting that the positive rate of a multipotent marker OCT4/TRA-1-60 is more than 90% in a flow mode, preparing single-cell suspension for standby after the hPSC is resolved into single cells, taking out an MSC pore plate for overnight culture, absorbing and discarding an original MSC amplification culture medium MH01, adding a multipotent stem cell amplification culture medium mTESR1, planting a proper amount of hiPSC single-cell suspension in a hole with hMSC as bottom cells, and adding 10 mu M of Y27632; 3) The second day after hPSC inoculation is marked as the beginning of the induction stage, marked as the 0 th day D0 of differentiation, old culture medium is sucked and abandoned into a hole containing hMSC and hiPSC, differentiation culture medium A is added, the differentiation culture medium A is sucked and abandoned when the first day D1 of induction differentiation is conducted, basic culture medium is replaced, hiPSC can be induced to mesodermal precursor cells when the induction of D1 is finished, differentiation culture medium B is added when the second day D2 of induction differentiation is conducted, fresh differentiation culture medium B is replaced every day, differentiation culture medium C is replaced on the fourth day of induction differentiation, differentiation culture medium C is replaced, the differentiation culture medium C is replaced every day, differentiation culture medium D is replaced on the sixth day of induction, the differentiation culture medium D is continuously induced for two days, the first stage is finished when the induction is finished, and large-area spontaneous beating myocardial cells can be seen.
  7. 7. An induction culture method for rapidly obtaining spontaneously beating myocardial balls according to claim 2, comprising the following stages: The second stage is a 3D culture stage, and the spontaneous beating myocardial cells induced in the first stage are digested and separated briefly by adopting an enzyme-free reagent and then are subjected to a pipette tip selecting treatment, so that the spontaneous beating myocardial balls are obtained.
  8. 8. The method for inducing and culturing myocardial balls capable of rapidly acquiring spontaneous beating according to claim 7, wherein the second stage comprises the steps of: 1) Taking out myocardial cells induced to D7, sucking and discarding old culture medium, cleaning with DPBS, adding 0.5mM EDTA digestion solution, incubating at room temperature, sucking and discarding EDTA digestion solution, adding myocardial cell maintenance culture medium, at this time, slightly stripping the clones along the clone edges in the orifice plate by a pipette gun head, transferring the stripped myocardial clones into an EP tube by a pipette, blowing and mixing uniformly, and standing on ice; 2) The cell suspension after standing can be seen to separate cells, the larger myocardial clone is sunk at the bottom, the upper layer is also provided with some other cells, the upper layer cells are sucked away, only the myocardial small mass at the bottom is left, the completely melted organoid matrigel is rapidly added on ice, after being blown and evenly mixed, the mixture of the myocardial mass and the organoid matrigel is split into a pore plate, each pore is added with the mixed solution of the myocardial mass and the organoid matrigel, when the mixture is added, a dome structure is formed by dripping from top to bottom, after the split charging is finished, the pore plate is solidified with matrigel, after the solidification is finished, each pore is added with myocardial cell maintenance culture medium, and the independent 3D myocardial balls capable of spontaneous beating can be obtained after 5 days of culture.

Description

Induction medium and method for rapidly obtaining spontaneously beating myocardial balls Technical Field The invention relates to the technical field of cell differentiation and the technical field of myocardial tissue regeneration medicine, in particular to an induction culture medium and a method for rapidly obtaining spontaneously beating myocardial balls. Background Human pluripotent stem cells (hpscs) include human induced pluripotent stem cells (hipscs) and human embryonic stem cells (hescs) that have the ability to differentiate into any somatic cell. Up to now hpscs have been shown to be powerful in the field of in vitro disease modeling, drug screening and developmental biology, and in the field of regenerative medicine, induced pluripotent stem cells (ipscs) derived islets obtained in vitro, neurons have been used in human clinical trials for the treatment of diabetes and epilepsy. The myocardial cells of mammals basically exit the cell cycle after birth, the myocardial cells are switched from a proliferation mode to a terminal differentiation mode, which means that human heart muscle becomes one of tissues with the worst regeneration capacity, when myocardial infarction occurs, damaged myocardial cells die largely, and the heart cannot regenerate new myocardial cells to replace, so that in order to solve the defect that the heart is not regenerated, scientists think that external functional myocardial cells can be transplanted to a heart injury area to directly replace dead cells when the heart is injured. The in vitro myocardial induction methods disclosed at present have several problems such as long in vitro induction time, complex culture medium composition (such as the patent of application numbers CN 202411392887 and CN 202311111730), or reduced induction time, but result in too low myocardial cell yield (such as the patent of application number CN 202510140533). In addition, the presently disclosed methods simply obtain cardiomyocytes earlier in development, which have insufficient maturity and poor performance compared to real cardiomyocytes in vivo. Therefore, in order to solve the above problems, it is important to provide a method which is simple and easy to operate and can greatly improve the in vitro yield and maturity of the cardiomyocytes derived from hpscs. Disclosure of Invention In order to solve the technical problems in the background art, the invention provides an induction culture medium and a method for rapidly obtaining a myocardial ball capable of spontaneously beating. The specific technical scheme is as follows: An induction culture medium for rapidly obtaining spontaneous beating myocardial balls comprises a basic culture medium, a differentiation culture medium A, a differentiation culture medium B, a differentiation culture medium C and a differentiation culture medium D; Wherein, the Basic culture medium, DMEM/F12 culture medium +50 μg/mL vitamin C +1XB27-insulin +100deg.X trace element B; Differentiation medium A basal medium+6mu.M Chir99021; differentiation Medium B basal Medium+2.5. Mu.M IWR1+2.5. Mu.M IWP2+4mM HCl; Differentiation medium C basal medium+4 mM hydrochloric acid; Differentiation medium D DMEM/F12 medium+50 μg/mL vitamin C+1XB27+100Xtrace element B. Preferably, the induction medium for rapidly obtaining spontaneously beating myocardial balls further comprises: Cardiomyocyte maintenance medium RPMI 1640 medium +1XB27+50. Mu.g/mL vitamin C +5ng/mL FGF2. An induction culture method for quickly obtaining spontaneously beating myocardial balls, which uses an induction culture medium for quickly obtaining spontaneously beating myocardial balls for induction culture. Preferably, the induction culture method for rapidly obtaining the spontaneously beating myocardial balls comprises the following steps: the first stage was to co-culture hiPSC and hMSC in 2D culture to obtain spontaneously beating cardiomyocytes. Preferably, the hiPSC has a starting cell amount of 5 ten thousand per square centimeter and the hMSC has a starting cell amount of 3 ten thousand per square centimeter. Preferably, the first stage comprises the steps of: 1) In the MSC plating stage, a stem cell matrigel coated cell culture plate is prepared in advance, hMSC from P3-P5 generation human umbilical cord separation sources is selected and planted in a coated pore plate, and MSC expansion culture medium MH01 is added for overnight culture; 2) The hPSC single-layer inoculation comprises the steps of selecting hiPSC with good state, detecting that the positive rate of a multipotent marker OCT4/TRA-1-60 is more than 90% in a flow mode, preparing single-cell suspension for standby after the hPSC is resolved into single cells, taking out an MSC pore plate for overnight culture, absorbing and discarding an original MSC amplification culture medium MH01, adding a multipotent stem cell amplification culture medium mTESR1, planting a proper amount of hiPSC single-cell suspension in a hole with hMSC