CN108660107A - A kind of skeletal muscle organoid construction method - Google Patents

A kind of skeletal muscle organoid construction method Download PDF

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CN108660107A
CN108660107A CN201810491120.4A CN201810491120A CN108660107A CN 108660107 A CN108660107 A CN 108660107A CN 201810491120 A CN201810491120 A CN 201810491120A CN 108660107 A CN108660107 A CN 108660107A
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skeletal muscle
mpcs
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CN108660107B (en
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邹晓晖
欧阳宏伟
刘怡孝
吴兵兵
安晟锐
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Zhejiang University ZJU
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    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
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Abstract

The present invention relates to a kind of skeletal muscle organoid construction method, this method includes：It is co-cultured and the step of obtain the class musculature block that can twitch after induction from the 3D in martrigel is detached into the RA cells (RACs) in flesh progenitor cells (MPCs) and its skeletal muscle in mice skeletal.The method of the present invention is simple, and operability is strong, and obtained skeletal muscle organoid can be shunk, and in conjunction with the physiological function of moveheat method quantitative analysis skeletal muscle organoids, is suitable for high-throughput medicine sieve research.

Description

A kind of skeletal muscle organoid construction method
Technical field
The present invention relates to a kind of construction methods of organoid, in particular it relates to which a kind of skeletal muscle organoid is built Method.
Background technology
In skeletal muscle regeneration research, 2D cultivating systems are most frequently used for expanding MPCs and induction MPCs is divided into myotube, But this cultural method cannot maintain for a long time, and the cultivating system of larger needs complexity is differed with natural muscle anatomical structure To cause contraction of muscle.Numerous studies find that in the regulation and control of muscle stem cell Micro-environmental cues, cell-ECM and cell-are extracellular Interaction between matrix plays an important role, however 2D cultivating systems have been not suitable for due to being limited by dimension for running business into particular one The research that born of the same parents-cell and cell-extracellular matrix interaction regulate and control cell.Even if the 3D cultured tissue engineering fleshes reported recently Meat, otherwise wherein seed cell used is sarcoblast system differs greatly with MPCs or be from neonate rat or adult bone The impure MPCs cells composition complicated component that flesh is isolated is not easy to control.It is contemplated that answering to Skeletal Muscle Cell constituent The 3D organizational project muscle cultural methods of polygamy, a kind of simulation natural muscle microenvironment newly are urgently developed.
Organoid technology is a kind of using mammal multipotential stem cell or the stem cell self in adult tissue source Characteristic build cell masses with many cells type of the 3D similar to internal microenvironment in vitro.There are many groups in recent years Knit organ organoid research report, these organoids with many cells type complexity with corresponding in-vivo tissue device Official's height is similar.This technology is to study the development of histoorgan, and regeneration and pathology provide ideal platform.
Invention content
The object of the present invention is to provide a kind of skeletal muscle organoid construction method and applications.It is external for research skeletal muscle regeneration Research provides the new research platform similar with internal anathrepsis height.
The technical solution adopted by the present invention is：
A kind of skeletal muscle organoid construction method, this method include：Detached from mice skeletal at flesh progenitor cells (myogenic progenitorcells, MPCs) and RA cells (the rapidly adhering in its skeletal muscle Cells, RACs) 3D is co-cultured and is obtained the step of class musculature block that can be twitched after induction in martrigel Suddenly.
Specifically, this method includes：
1) by RACs and MPCs amplification in vitros to 3~4 generations；
2) RACs and MPCs are digested to cell suspension respectively with pancreatin and counted respectively；
3) required cell suspension is taken, RACs and MPCs is pressed 1:1 number removes supernatant than mixing, centrifugation；
4) cell mixing is resuspended with matrigel after dilution, puts later on ice；
5) matrigel cell re-suspension liquids are drawn, are dripped in culture dish, culture dish inversion is put into CO later2In incubator 15min makes matrigel solidify；
6) the matrigel cell mixtures after solidification are detached from culture dish, are put into growth medium (Growth Medium, GM) inner suspension culture is for 24 hours；
7) matrigel cell mixtures are transferred to differential medium (Differentiation from growth medium Medium, DM) culture 14 days to get.
Described MPCs, the RACs is prepared by the method included the following steps：
1) Hind limb gastrocnemius and tibialis anterior of broken vertebra execution and the C57BL/6 male mices of sterilization treatment are taken；Use HBSS Clean 3 times and shave tendon, fat and fascia；
2) muscle is cut into meat slurry with eye scissors；Centrifugation, removes supernatant；It is washed with HBSS and is centrifuged again；
3) supernatant is removed, adds 0.2% clostridiopetidase A Ⅺ after 37 DEG C of preheatings that muscle is resuspended and starts to digest, 1 is incubated in 37 DEG C of water-baths Hour, wherein it is primary to overturn mixing per 10min；
4) it after supernatant is removed in meat slurry-enzymatic mixture centrifugation, is resuspended with diapase solution, is incubated 1 hour in 37 DEG C of water-baths；
5) meat slurry-enzymatic mixture centrifugation is resuspended with 0.1% pancreatin after removing supernatant, 45min is incubated in 37 DEG C of water-baths；
6) it centrifuges, is resuspended with GM after removing supernatant；
7) object is resuspended with 70 μm of aperture cell sieve filterings；
8) re-suspension liquid is aspirated with syringe；
9) re-suspension liquid is transferred in the culture dish after Collagen I coating, is named as PP1；
10) supernatant is transferred in new Collagen I coating 60mm culture dishes and is ordered after placing 2h in 37 DEG C of cell incubators Fresh growth medium (Growth Medium, GM) is added in entitled PP2 in PP1；
11) supernatant in PP2 is transferred in new Collagen I coating culture dish afterwards for 24 hours and is named as PP3, added in PP2 Enter fresh culture；
12) it repeats step (11) and inhales the culture medium abandoned and renewed after 72h again in PP6 culture dishes until obtaining PP6, supernatant；
Cell in PP1, PP2 is RA cells (Rapidly adhering cells, RACs)；In PP3, PP4 Cell, at flesh progenitor cells, i.e. MPCs after purification amplification.
The present invention also provides a kind of Moveheat to detect skeletal muscle organoid method, and this method comprises the following steps：
1) the ripe skeletal muscle organoid of culture is taken out from incubator, the video of its contraction is taken under 40X mirrors (about 1min)；
2) after, obtaining video, using each frame image in the averaging operator processing video of 15*15, mould is carried out to image Gelatinization；
3) gray scale difference of n+3 frames and n-th frame respective pixel, is calculated；
4) average value of each pixel grey scale variation of all frames, the temperature as corresponding region image change, are finally asked.
Advantageous effect possessed by the present invention：
The method of the present invention is simple, and operability is strong, and obtained skeletal muscle organoid can be shunk, in conjunction with moveheat methods The physiological function of quantitative analysis skeletal muscle organoid is suitable for high-throughput medicine sieve research.
Description of the drawings
Fig. 1 is the result of separation and the identification of Muscle progenitor cells.
Wherein A, the MPCs under the white light visual field.B, MYOD1 and PAX7 immunofluorescence contaminate altogether.C, Muscle progenitor cells account for total cell Number ratio.
By to PAX7 in immunofluorescence results in Fig. 1+MYOD1+,PAX7-MYOD1+,PAX7+MYOD1-Cell number accounts for The ratio of total cell number, which calculates, understands that isolated purity is higher at flesh progenitor cells.
Fig. 2 is that RACs can promote MPCs at flesh in skeletal muscle organoid incubation
R in Fig. 2：The independent 3D cultures of RACs；MR：RACs and MPCs 3D are co-cultured；M：The independent 3D cultures of MPCs.A：Body formula Skeletal muscle organoid under mirror.B：Skeletal muscle organoid under 40X mirrors.C：MHC (myosin heavy chain) immunofluorescence.
Isolated RACs from mice skeletal and MPCs is co-cultured, is cultivated 1 day in growth medium, at flesh The skeletal muscle (Fig. 2, MR group) that can be twitched is obtained after being cultivated in inducing culture 2 weeks, however under identical condition of culture RACs and MPCs is respectively individually cultivated, and will not form the skeletal muscle organoid (Fig. 2, R group and M groups) that can be twitched, this shows RACs promotes MPCs myogenic differentiations to play a key effect in bone in organoid incubation.Fig. 3 is from mice skeletal Detach RA cells (RACs) and at flesh progenitor cells (MPCs) flow chart.
As shown, cell adherent in 24 hours in the postdigestive suspension of muscle is known as RA cells by us The adherent time is longer than 24 hours cells and is known as slow attached cell (slowly by (rapidly adhering cells, RACs) adhering cells,SACs)
Fig. 4 is skeletal muscle organoid culture flow diagram.
Specific implementation mode
The present invention is further explained in the light of specific embodiments, but does not limit protection scope of the present invention.
The separation and identification of 1.MPCs and RA cells.
1.1 skeletal muscle RA cells and MPCs separation
1) 37 DEG C preheating clostridiopetidase A Ⅺ (C7657, Sigma-Aldrich), dispase II ((D4693-1G, Sigma) and Pancreatin；
2) the big C57BL/6 male mices of two 6~8w are broken after vertebra is put to death and is put into 75% alcohol the 10min that sterilizes；
3) Hind limb gastrocnemius and tibialis anterior are taken, 3 times are cleaned with HBSS and shaves tendon, fat and fascia；
4) muscle is cut into meat with eye scissors and starches (about 1mm3)；
5) meat slurry is transferred in 15ml centrifuge tubes；Centrifugation；
6) supernatant is removed, washed with HBSS (24020-117, Invitrogen) and is centrifuged again；
7) supernatant is removed, adds 0.2% clostridiopetidase A, Ⅺ 10ml after 37 DEG C of preheatings that muscle is resuspended and starts to digest, in 37 DEG C of water-baths It incubates 1 hour, wherein it is primary to overturn mixing per 10min；
8) meat slurry -4 DEG C of enzymatic mixture 930g centrifuges 5min, is resuspended with 10ml diapase solution after removing supernatant, 37 DEG C of water It is incubated in bath 1 hour, wherein it is primary to overturn mixing per 10min；
9) meat slurry -4 DEG C of enzymatic mixture 930g centrifuges 5min, is resuspended with 0.1% pancreatin of 10ml after removing supernatant, 37 DEG C of water-baths 45min is incubated in pot, it is primary (soft) per 10min mixings；
10) 4 DEG C of 930g centrifuge 5min, are resuspended with 10ml GM after removing supernatant；
11) object is resuspended with 70 μm of aperture cell sieve filterings；
12) successively respectively with 18G, 23G and 27G10ml syringes aspirate re-suspension liquid (each 2 times)；
13) re-suspension liquid is transferred in the 60mm culture dishes after Collagen I coating, is named as PP1；
14) supernatant is transferred in new Collagen I coating 60mm culture dishes and is ordered after placing 2h in 37 DEG C of cell incubators 3ml fresh growth mediums (Growth Medium, GM) are added in entitled PP2 in PP1；
Growth medium (Growth Medium, GM)：H-DMEM, 10%FBS, 10%HS, 0.5%CEE, penicillin (100U/ml), streptomysin (100U/ml).
15) supernatant in PP2 is transferred in new Collagen I coating 60mm culture dishes afterwards for 24 hours and is named as PP3, in PP2 Middle addition 3ml fresh cultures；
16) it repeats step (15) and inhales the culture medium abandoned and renewed after 72h again in PP6 culture dishes until obtaining PP6, supernatant.
Cell in PP1, PP2 is RA cells (Rapidly adhering cells, RACs), cellular morphology with Fibroblast is very much like；Cell in PP3, PP4 is about largely muscle stem cell, at flesh ancestral after purification amplification Cell, i.e. MPCs.
MPCs purification steps：
1. culture medium is abandoned in suction, washed with 2ml1X PBS；
2. with 0.05% pancreatin of 0.5ml, 37 DEG C of digestion 30s, later plus 0.5mlGM terminates digestion；
3. the cell under digestion, which is transferred to 930g in 15ml centrifuge tubes, centrifuges 5min；
4. supernatant is abandoned in suction, 3mlGM is added, cell is resuspended and cell suspension is transferred to the new coated culture of 60mm Collagen Is In ware, culture dish is then put into adherent 30min in incubator；
5. after the adherent processing of 30min, non-attached cell in supernatant to be transferred to the new coated culture dish of 60mm Collagen Is In.
So far primary purifying is completed, purifies the number of repetition depending on fibroblast-like cells severity of mixing up.
1.2 MPCs are identified
It is to express after muscle stem cell activation into flesh transcription factor that PAX7, which is muscle stem cell classics marker, MYOD1, Flesh progenitor cells classical marker, therefore we identify the muscle ancestral cells being separated to both marker.Such as The middle muscle ancestral cells proportion of isolated muscle ancestral cells shown in Fig. 1 is about 83%.
2. the culture of skeletal muscle organoid
1) growth medium and matrigel shift to an earlier date 30min and are pre-chilled on ice；
2) continue to be pre-chilled on ice after matrigel being diluted 1 times with growth medium according to use demand amount；
3) by RACs and MPCs amplification in vitros to 3~4 generations, RACs and MPCs is enable respectively to reach at least up to 7*105；
4) RACs and MPCs are digested to cell suspension respectively with pancreatin and counted respectively；
5) dosage (RACs of RACs and MPCs is determined according to the number (R, RM, M every group three) for making organoid: MPCs=1:1) required cell suspension (R groups 2.8*10, is then taken5Each 1.4*10 of a RACs, RM group RACs and MPCs5It is a, M groups MPCs 2.8*105It is a), RACs and MPCs is pressed 1:1 number removes supernatant than mixing, 200g after centrifuging 5min.
6) cell mixing is resuspended in every group of GM with 35 μ l volumes, puts 5min is pre-chilled on ice later, then every group of 35 μ l of addition The matrigel of volume precooling.
7) matrigel cell re-suspension liquids are drawn with 200 μ l pipette tips after precooling, carefully drips to (20 μ l/ drops) 60mm cultures In ware, culture dish inversion is put into CO later215min makes matrigel solidify in incubator.
8) the matrigel cell mixtures after solidification are detached from 1ml big pipette tips in culture dish, are put into grown cultures Ji Li suspends culture for 24 hours.
Growth medium (Growth Medium, GM)：H-DMEM, 10%FBS, 10%HS, 0.5%CEE, penicillin (100U/ml), streptomysin (100U/ml).
9) matrigel cell mixtures are transferred to differential medium (Differentiation from growth medium Medium, DM) it cultivates 14 days.
Induction culture medium (differentiation medium, DM)；H-DMEM, 2%HS, penicillin (100U/ Ml), streptomysin (100U/ml).
Skeletal muscle organoid physiological function detects
It is about 2.5mm to cultivate obtained skeletal muscle organoid diameter, not easy-to-use existing muscle physiological Function detection means Detection, in order to detect skeletal muscle organoid physiological function, we have invented Moveheat detection methods.
Moveheat detects skeletal muscle organoid principle：
1) the ripe skeletal muscle organoid of culture is taken out from incubator, the video of its contraction is taken under 40X mirrors (about 1min)；
2) after obtaining video, using each frame image in the averaging operator processing video of 15*15, image is obscured Change；
3) gray scale difference of n+3 frames and n-th frame respective pixel is calculated；
4) average value of each pixel grey scale variation of all frames, the temperature as corresponding region image change are finally asked.

Claims (4)

1. a kind of skeletal muscle organoid construction method, it is characterised in that：This method includes：Detached from mice skeletal at flesh The 3D in martrigel is co-cultured and through induction progenitor cells (MPCs) with the RA cells (RACs) in its skeletal muscle The step of obtaining the class musculature block that can be twitched afterwards.
2. a kind of skeletal muscle organoid construction method according to claim 1, it is characterised in that：This method includes：
1) by RACs and MPCs amplification in vitros to 3~4 generations；
2) RACs and MPCs are digested to cell suspension respectively with pancreatin and counted respectively；
3) required cell suspension is taken, RACs and MPCs is pressed 1:1 number removes supernatant than mixing, centrifugation；
4) cell mixing is resuspended with matrigel after dilution, puts later on ice；
5) matrigel cell re-suspension liquids are drawn, are dripped in culture dish, culture dish inversion is put into CO later215min in incubator Matrigel is set to solidify；
6) the matrigel cell mixtures after solidification are detached from culture dish, are put into growth medium (Growth Medium, GM) inner suspension culture is for 24 hours；
7) matrigel cell mixtures are transferred to differential medium (Differentiation from growth medium Medium, DM) culture 14 days to get.
3. a kind of skeletal muscle organoid construction method according to claim 1 or 2, it is characterised in that：The MPCs, RACs is prepared by the method included the following steps：
1) Hind limb gastrocnemius and tibialis anterior of broken vertebra execution and the C57BL/6 male mices of sterilization treatment are taken；3 are cleaned with HBSS All over and shave tendon, fat and fascia；
2) muscle is cut into meat slurry with eye scissors；Centrifugation, removes supernatant；It is washed with HBSS and is centrifuged again；
3) supernatant is removed, adds 0.2% clostridiopetidase A Ⅺ after 37 DEG C of preheatings that muscle is resuspended and starts to digest, incubated in 37 DEG C of water-baths 1 hour, It is primary that mixing is wherein overturned per 10min；
4) it after supernatant is removed in meat slurry-enzymatic mixture centrifugation, is resuspended with diapase solution, is incubated 1 hour in 37 DEG C of water-baths；
5) meat slurry-enzymatic mixture centrifugation is resuspended with 0.1% pancreatin after removing supernatant, 45min is incubated in 37 DEG C of water-baths；
6) it centrifuges, is resuspended with GM after removing supernatant；
7) object is resuspended with 70 μm of aperture cell sieve filterings；
8) re-suspension liquid is aspirated with syringe；
9) re-suspension liquid is transferred in the culture dish after Collagen I coating, is named as PP1；
10) supernatant is transferred in new Collagen I coating 60mm culture dishes and is named as after placing 2h in 37 DEG C of cell incubators Fresh growth medium (Growth Medium, GM) is added in PP2 in PP1；
11) supernatant in PP2 is transferred in new Collagen I coating culture dish afterwards for 24 hours and is named as PP3, be added in PP2 new Fresh culture medium；
12) it repeats step (11) and inhales the culture medium abandoned and renewed after 72h again in PP6 culture dishes until obtaining PP6, supernatant；
Cell in PP1, PP2 is RA cells (RACs)；Cell in PP3, PP4, at flesh after purification amplification Progenitor cells, i.e. MPCs.
4. a kind of Moveheat detects skeletal muscle organoid method, it is characterised in that：This method comprises the following steps：
1) the ripe skeletal muscle organoid of culture is taken out from incubator, the video of its contraction is taken under 40X mirrors；
2) after, obtaining video, using each frame image in the averaging operator processing video of 15*15, image is blurred；
3) gray scale difference of n+3 frames and n-th frame respective pixel, is calculated；
4) average value of each pixel grey scale variation of all frames, the temperature as corresponding region image change, are finally asked.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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