CN108660107B

CN108660107B - Skeletal muscle organoid construction method

Info

Publication number: CN108660107B
Application number: CN201810491120.4A
Authority: CN
Inventors: 邹晓晖; 欧阳宏伟; 刘怡孝; 吴兵兵; 安晟锐
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-05-21
Filing date: 2018-05-21
Publication date: 2021-04-13
Anticipated expiration: 2038-05-21
Also published as: CN108660107A

Abstract

The invention relates to a skeletal muscle organoid construction method, which comprises the following steps: 3D coculture of myoblasts isolated from mouse skeletal Muscle (MPCs) and Rapidly Adherent Cells (RACs) in skeletal muscle in martigel and differentiation induction to obtain twitch-like muscle tissue mass. The method is simple and easy, has strong operability, can be used for obtaining the skeletal muscle organoid capable of contracting, is combined with the moveheat method to quantitatively analyze the physiological function of the skeletal muscle organoid, and is suitable for high-throughput drug screening research.

Description

Skeletal muscle organoid construction method

Technical Field

The invention relates to a construction method of a similar organ, in particular to a construction method of a skeletal muscle similar organ.

Background

In skeletal muscle regeneration studies, 2D culture systems are most often used to expand MPCs and induce MPCs to differentiate into myotubes, but this culture method does not allow long-term maintenance, and requires a complicated culture system to initiate muscle contraction, which is significantly different from the natural muscle anatomy. Numerous studies have found that cell-cell and cell-extracellular matrix interactions play an important role in the signal regulation of the muscle stem cell microenvironment, however, the 2D culture system is not suitable for the study of cell-cell and cell-extracellular matrix interactions on cell regulation due to the dimensional limitation. Even in the recently reported 3D cultured tissue engineered muscles, the seed cells used therein are either myoblast cell lines with large differences from MPCs, or impure MPCs isolated from neonatal rat or adult skeletal muscle with a complex and poorly controllable cell composition. Therefore, considering the complexity of the components of skeletal muscle cells, a new 3D tissue engineering muscle culture method simulating the natural muscle microenvironment is in need of development.

Organoid technology is a cellular mass with multiple cell types that uses the properties of mammalian pluripotent stem cells or adult tissue-derived stem cell self-organization to construct 3D in vitro-like microenvironments. Organoid studies of various tissue organs have been reported in recent years, and these organoids with the complexity of multiple cell types have been highly similar to the corresponding in vivo tissue organs. The technology provides an ideal platform for researching the development, regeneration and pathology of tissues and organs.

Disclosure of Invention

The invention aims to provide a skeletal muscle organoid construction method and application. Provides a new research platform which is highly similar to the in vivo muscle regeneration for the research of skeletal muscle regeneration in vitro.

The technical scheme adopted by the invention is as follows:

a skeletal muscle organoid construction method, comprising: the method comprises the steps of co-culturing Myogenic Progenitor Cells (MPCs) separated from skeletal muscle of a mouse and Rapidly Adherent Cells (RACs) in skeletal muscle of the MPCs in martrigel in 3D, and obtaining a twitch-like muscle tissue block after differentiation induction.

Specifically, the method comprises the following steps:

1) amplifying RACs and MPCs in vitro to 3-4 generations;

2) respectively digesting the RACs and the MPCs into cell suspensions by pancreatin and respectively counting;

3) taking the required cell suspension, mixing RACs and MPCs according to the number ratio of 1:1, and centrifuging to remove the supernatant;

4) resuspending the cells with diluted matrigel, mixing well, and then placing on ice;

5) the matrigel cell resuspension was aspirated, dropped into a petri dish, and the dish was inverted and placed in CO₂Solidifying matrigel in an incubator for 15 min;

6) separating the solidified matrigel cell mixture from the culture dish, and putting the solidified matrigel cell mixture into a Growth Medium (GM) for suspension culture for 24 hours;

7) the matrigel cell mixture was transferred from the growth Medium to a Differentiation Medium (DM) and cultured for 14 days.

The MPCs and the RACs are prepared by the method comprising the following steps:

1) taking hind limb gastrocnemius and tibialis anterior of C57BL/6 male mice which have been sacrificed and sterilized by vertebral fracture; HBSS washing for 3 times and removing tendon, fat and fascia;

2) shearing the muscle into meat pulp by using an ophthalmic scissors; centrifuging and removing supernatant; washed with HBSS and centrifuged again;

3) removing supernatant, adding 37 deg.C preheated 0.2% collagenase XI, resuspending muscle, incubating in 37 deg.C water bath for 1 hr, and mixing by reversing every 10 min;

4) centrifuging the meat pulp-enzyme mixture to remove supernatant, then resuspending the mixture by using diapase solution, and incubating the mixture in a water bath at 37 ℃ for 1 hour;

5) centrifuging the meat pulp-enzyme mixture, removing supernatant, resuspending with 0.1% pancreatin, and incubating at 37 deg.C for 45 min;

6) centrifuging, removing supernatant, and then resuspending with GM;

7) filtering the heavy suspension by using a cell sieve with the aperture of 70 mu m;

8) aspirating the resuspension with a syringe;

9) transferring the heavy suspension into a culture dish coated with collagen I, and naming the culture dish as PP 1;

10) after being placed in a cell culture box at 37 ℃ for 2 hours, the supernatant was transferred to a new collagen I-coated 60mm petri dish and named PP2, and a fresh Growth Medium (grown Medium, GM) was added to PP 1;

11) after 24h, the supernatant from PP2 was transferred to a new collagen I coated petri dish and named PP3, and fresh medium was added to PP 2;

12) repeating the step (11) until PP6 is obtained, and sucking and replacing new culture medium after supernatant is put in a PP6 culture dish for 72 hours;

cells in PP1, PP2 were Rapidly adherent cells (racldly attaching cells, RACs); cells in PP3, PP4, were purified and expanded in vitro to give myoblasts, i.e., MPCs.

The invention also provides a method for detecting skeletal muscle organoids by Moveheat, which comprises the following steps:

1) taking out the cultured mature skeletal muscle organs from the incubator, and shooting a contracted video (about 1min) under a 40X mirror;

2) after the video is obtained, processing each frame of image in the video by using an average operator of 15 × 15, and blurring the image;

3) calculating the gray difference of the pixels corresponding to the n +3 frame and the nth frame;

4) and finally, calculating the average value of the gray level change of each pixel of all frames as the heat of the image change of the corresponding area.

The invention has the following beneficial effects:

the method is simple and easy, has strong operability, can be used for obtaining the skeletal muscle organoid capable of contracting, is combined with the moveheat method to quantitatively analyze the physiological function of the skeletal muscle organoid, and is suitable for high-throughput drug screening research.

Drawings

FIG. 1 shows the results of the isolation and identification of muscle progenitor cells.

Where A, MPCs under white light field of view. B, MYOD1 and PAX7 immunofluorescent co-staining. C, the proportion of muscle progenitor cells to the total cell number.

By comparing the results of immunofluorescence in FIG. 1 with that of PAX7⁺MYOD1⁺,PAX7^-MYOD1⁺,PAX7⁺MYOD1^-The ratio of the cell number to the total cell number is calculated, so that the myoblast progenitor cells with high purity can be obtained by separation.

FIG. 2 shows that RACs can promote the formation of MPCs into muscle during the skeletal muscle organoid culture process

R in FIG. 2: 3D culture of RACs alone; MR: co-culturing RACs and MPCs in 3D; m: MPCs were 3D cultured alone. A: the skeletal muscle organoid under the body scope. B: the skeletal muscle organoid was under a 40X scope. C: MHC (myostatin heavy chain) immunofluorescence.

RACs and MPCs separated from mouse skeletal muscle are co-cultured, cultured in a growth medium for 1 day, and cultured in a myogenic induction medium for 2 weeks to obtain skeletal muscle capable of twitching (figure 2, MR group), however, RACs and MPCs are independently cultured under the same culture condition to form skeletal muscle organs capable of twitching (figure 2, R group and M group), which shows that RACs play a key role in promoting the myogenic differentiation of MPCs in the process of culturing the skeletal organs. FIG. 3 is a flow chart of the isolation of Rapidly Adherent Cells (RACs) and Myoblasts (MPCs) from mouse skeletal muscle.

As shown in the figure, cells attached within 24 hours of the muscle digestion suspension are called fast attached cells (RACs), and cells attached for longer than 24 hours are called Slow Attached Cells (SACs)

FIG. 4 is a schematic diagram of a skeletal muscle organoid culture process.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Isolation and identification of MPCs and rapidly adherent cells.

1.1 Rapid isolation of adherent cells from skeletal muscle and MPCs

1) Collagenase XI (C7657, Sigma-Aldrich), dispase II ((D4693-1G, Sigma) and pancreatin preheated at 37 ℃;

2) two 6-8 w large C57BL/6 male mice are killed by vertebral column breakage and then sterilized in 75% alcohol for 10 min;

3) taking hind limb gastrocnemius and tibialis anterior, washing with HBSS for 3 times, and removing tendon, fat and fascia;

4) shearing muscle into pulp (about 1 mm) with ophthalmic scissors³)；

5) Transferring the meat pulp into a 15ml centrifuge tube; centrifuging;

6) the supernatant was removed, washed with HBSS (24020-117, Invitrogen) and centrifuged again;

7) removing supernatant, adding 10ml of preheated 37 deg.C 0.2% collagenase XI, resuspending muscle, incubating in 37 deg.C water bath for 1 hr, and mixing by reversing every 10 min;

8) centrifuging the meat pulp-enzyme mixture at 930g for 5min at 4 ℃, removing supernatant, then resuspending with 10ml diapase solution, incubating in a water bath kettle at 37 ℃ for 1 h, and reversing and mixing uniformly every 10 min;

9) centrifuging the meat pulp-enzyme mixture at 930g for 5min at 4 deg.C, removing supernatant, re-suspending with 10ml of 0.1% pancreatin, incubating in water bath at 37 deg.C for 45min, and mixing uniformly every 10min (gently);

10) centrifuging at 4 deg.C for 5min at 930g, removing supernatant, and resuspending with 10ml GM;

11) filtering the heavy suspension by using a cell sieve with the aperture of 70 mu m;

12) aspirating the resuspension sequentially (2 times each) using syringes with 18G, 23G and 27G10ml, respectively;

13) transferring the heavy suspension into a collagen I coated 60mm culture dish, and naming the dish as PP 1;

14) after 2h at 37 ℃ in the cell culture chamber, the supernatant was transferred to a new collagen I-coated 60mm petri dish and named PP2, 3ml of fresh Growth Medium (grown Medium, GM) was added to PP 1;

growth Medium (GM): H-DMEM, 10% FBS, 10% HS, 0.5% CEE, penicillin (100U/ml), streptomycin (100U/ml).

15) After 24h the supernatant from PP2 was transferred to a new collagen I coated 60mm petri dish and named PP3, to which 3ml of fresh medium was added in PP 2;

16) and (5) repeating the step (15) until PP6 is obtained, and sucking and replacing new culture medium after supernatant is put in a PP6 culture dish for 72 hours.

Cells in PP1 and PP2 are Rapidly adherent cells (racldly attaching cells, RACs), and the morphology of the cells is very similar to that of fibroblasts; PP3, PP4, has approximately the majority of myocytes as muscle stem cells, and after in vitro purification and expansion, myoblasts, i.e., MPCs, are obtained.

In-vitro purification of MPCs:

1. the medium was aspirated off and washed with 2ml of 1X PBS;

2. digesting with 0.5ml of 0.05% pancreatin at 37 ℃ for 30s, and then adding 0.5ml GM to terminate the digestion;

3. transferring the digested cells to a 15ml centrifuge tube, and centrifuging for 5min at 930 g;

4. sucking and removing the supernatant, adding 3ml of GM (granulocyte macrophage colony stimulating factor) heavy suspension cells, transferring the cell suspension into a new culture dish coated with 60mm collagen I, and then putting the culture dish into an incubator to adhere to the wall for 30 min;

after 5.30 min adherent treatment, the non-adherent cells in the supernatant were transferred to a new 60mm collagen i coated petri dish.

This is done once and the number of purification repetitions depends on the degree of fibroblast contamination.

1.2 identification of MPCs

PAX7 is a classic marker of muscle stem cells, MYOD1 is a classic marker of myogenic transcription factors expressed after muscle stem cell activation, i.e., myogenic progenitor cells, so we used these two markers to identify isolated muscle stem/progenitor cells. The muscle stem/progenitor cells isolated as shown in FIG. 1 account for approximately 83% of the total muscle stem/progenitor cells.

2. Culture of skeletal muscle organoids

1) Pre-cooling the growth medium and matrigel on ice 30min in advance;

2) diluting matrigel by 1 time with a growth medium according to the using demand, and then continuing to pre-cool on ice;

3) amplifying RACs and MPCs to 3-4 generations in vitro, so that the RACs and the MPCs can reach at least 7 x 10 respectively⁵；

4) Respectively digesting the RACs and the MPCs into cell suspensions by pancreatin and respectively counting;

5) the amounts of RACs and MPCs were determined based on the number of organoids prepared (R, RM, M three in each group) (RACs: MPCs ═ 1:1) and the desired cell suspension was taken (R group 2.8 × 10)⁵1.4 × 10 each of RACs, RM groups RACs and MPCs⁵M group of MPCs 2.8 x 10⁵Individually), RACs and MPCs were mixed at a ratio of 1:1, centrifuged at 200g for 5min and the supernatant removed.

6) Each group was mixed well with a 35. mu.l volume of GM resuspended cells, then chilled on ice for 5min, and then 35. mu.l volume of chilled matrigel was added to each group.

7) The matrigel cell resuspension was aspirated with a pre-cooled 200. mu.l pipette tip, carefully dropped (20. mu.l/drop) into a 60mm petri dish, which was then placed upside down in CO₂The matrigel was coagulated in an incubator for 15 min.

8) The solidified matrigel cell mixture was detached from the petri dish using a 1ml large tip and placed in growth medium for suspension culture for 24 h.

9) Matrigel cell mixtures were transferred from growth Medium to Differentiation Medium (DM) for 14 days.

Differentiation induction medium (DM); H-DMEM, 2% HS, penicillin (100U/ml), streptomycin (100U/ml).

Skeletal muscle organoid physiological function detection

The diameter of the skeletal muscle organoid obtained by culture is about 2.5mm, the detection is not easy to use the existing muscle physiological function detection means, and the Moveheat detection method is invented for detecting the physiological function of the skeletal muscle organoid.

Moveheat detects skeletal muscle organoid principle:

1) taking the cultured mature skeletal muscle organoids out of the incubator, and taking a video of contraction thereof under a 40X mirror (about 1 min);

Claims

1. A skeletal muscle organoid construction method is characterized in that: the method comprises the following steps:

1) amplifying RACs and MPCs in vitro to 3-4 generations;

6) separating the solidified matrigel cell mixture from the culture dish, and putting the mixture into a growth medium for suspension culture for 24 hours;

7) transferring the matrigel cell mixture from a growth medium to a differentiation medium for 14 days to obtain the matrigel cell mixture;

6) centrifuging, removing supernatant, and then resuspending with fresh growth medium;

8) aspirating the resuspension with a syringe;

10) after being placed in a cell culture box at 37 ℃ for 2 hours, the supernatant is transferred to a new collagen I coated 60mm culture dish and named as PP2, and a fresh growth medium is added into PP 1;

12) repeating the step (11) until PP6 is obtained, and sucking and replacing new culture medium after supernatant is put in a PP6 culture dish for 72 hours; cells in PP1, PP2 were fast adherent cells (RACs); the cells in PP3 and PP4 are purified and expanded in vitro to become myoblast progenitor cells, namely MPCs;

growth medium: H-DMEM, 10% FBS, 10% HS, 0.5% CEE, penicillin: 100U/ml, streptomycin: 100U/ml;

a differentiation medium; H-DMEM, 2% HS, penicillin: 100U/ml, streptomycin: 100U/ml.