CN114250199A - Method and medium for culturing spine-spinal cord organoid - Google Patents

Method and medium for culturing spine-spinal cord organoid Download PDF

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CN114250199A
CN114250199A CN202111372401.6A CN202111372401A CN114250199A CN 114250199 A CN114250199 A CN 114250199A CN 202111372401 A CN202111372401 A CN 202111372401A CN 114250199 A CN114250199 A CN 114250199A
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culture medium
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spinal cord
spine
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CN114250199B (en
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项鹏
李伟强
李岱睿
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Sun Yat Sen University
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Abstract

The invention discloses a method for culturing spine-spinal cord organoid and a culture medium, the invention utilizes a composition in the culture medium for culturing the spine-spinal cord organoid, the culture medium induces and utilizes neuromesoderm progenitor cells to obtain the spine-spinal cord organoid, not only provides a complete in vitro model for the early development of the spine and the spinal cord, but also provides a theoretical basis for clarifying the normal development mechanism of the spine and the spinal cord and exploring the pathogenesis, treatment target and prevention and treatment means of related diseases.

Description

Method and medium for culturing spine-spinal cord organoid
Technical Field
The invention relates to the technical field of organoids, in particular to a culture method and a culture medium for spinal-spinal organoids.
Background
The spine and spinal cord are the main components of the trunk. The spine is composed of a plurality of vertebrae, has high hardness and has certain mobility, so that the weight of the vast majority of the human body can be loaded, and the movement of the human body in a certain range can be guaranteed. A longitudinal hollow vertebral canal is formed inside the vertebral column from top to bottom to protect the spinal cord contained therein. The spinal cord is an important component of the central nervous system and is capable of transmitting signals from the brain to various parts of the body.
Dysplasia in either of the spinal column and spinal cord can lead to disease, and commonly includes spina bifida and congenital scoliosis. The incidence of these diseases is high and the pathogenesis is complex. The current treatment means has limited curative effect. Related Animal Models have been reported for the dysplasia of spinal cord, but these Models often have problems of insufficient simulation degree due to species differences (Ouellet J, Oden T. Animal Models for spinal cord research: state of the art, current conditions and future empirical applications [ J ]. European spinal cord journel, 2013,22(2): 81-95; Bobyn J D, Little D G, Gray R, et al. Animal Models for spinal cord J ]. Journal of regional research,2015,33(4): Skyn 467; upper I, ribbon J. Animal Models for evaluating spinal cord Defects J. 157: 157). Therefore, establishing an effective human early-stage spinal column and spinal cord development model has important significance for clarifying normal development mechanism of spinal column and spinal cord, and exploring pathogenesis and prevention and treatment means of related diseases.
To construct a complete in vitro model, the developmental origin of the spine and spinal cord is first defined. In the early stages of gastrulation, the caudal epiblast region and the primitive streak region present a population of Axial Stem Cells (ASCs) capable of growing somatic tissues to extend the embryo axially (Cambray N, Wilson V.axial precursors with extended pore area to the motor nuclear expression [ J ]. Development,2002,129(20): 4855-. The precursors of the spine and spinal cord are important parts of the anterior-posterior axis of the embryo. Among them, the spine develops mainly from the paraxial mesoderm, and the spinal cord develops mainly from the posterior neural tube. It was found by cell tracking experiments that the origin of the paraxial mesoderm and the posterior neural tube is not independent, but are derived from a specific somatic progenitor cell: neuromesodermal progenitors (NMPs) (Henrique D, Abranches E, Verrier L, et al. neurodermal progenitors and the labeling of the spinal cord [ J ] Development,2015,142(17): 2864-). NMPs have a bidirectional differentiation ability, and in vivo, they can differentiate into tissues such as bones and muscles via the somites, and also into the spinal cord via the neural tube. Based on the differentiation characteristics of NMPs, the NMPs can be used as high-quality seed cells for spinal cord development simulation and related disease pathogenesis exploration.
However, due to the short time and small amount of NMPs existing in vivo and the ethical constraints faced by human embryo research, NMPs cannot be isolated directly from the body. Pluripotent Stem Cells (PSCs), including Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs), have the ability to rapidly proliferate and differentiate in vitro, and they have been demonstrated to be able to induce differentiation into almost all cell types of each germ layer, which makes it possible to induce differentiation of NMPs by pluripotent stem cells in vitro.
Meanwhile, diseases caused by spinal cord dysplasia are usually the change of three-dimensional structures of tissues and organs, so that the traditional adherent cell culture mode cannot well simulate the diseases. The rapid development of organoid technology in recent years has enabled the three-dimensional simulation of the development of tissue and organs. In vitro organoid models have been successfully established for a variety of tissues, including early torso-like tissues constructed based on mouse NMPs (Veenvliot J V, Bolondi A, Kretzmer H, et al. mouse organizing cells self-organization into trunk-like structures with neural tubes and sources [ J ]. Science,2020,370 (6522)).
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a culture method and a culture medium for spinal-spinal organoids.
The first purpose of the invention is to provide a culture medium for spinal-spinal organoids.
The second purpose of the invention is to provide a method for culturing spinal-spinal cord organoids.
The third purpose of the invention is to provide the application of the culture medium in the culture of spine-spinal cord organoids.
The fourth purpose of the invention is to provide the spine-spinal cord organoid cultured by the culture method
A fourth object of the invention is the use of said spine-spinal cord organoids as a model for early development of the spine and/or spinal cord.
The invention therefore claims a culture medium for spinal-spinal organoids, comprising one or more of the culture media 1 to 3,
wherein, the culture medium 1 contains bFGF and EGF;
culture medium 2 contains insulin-transferrin-selenium supplement ITS, retinoic acid RA, SAG, glial cell line-derived neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, insulin-like growth factor IGF-I, bone morphogenetic protein BMP-4, TGF beta 1, L-ascorbic acid AA, dibutyl CAMP, beta-glycerophosphate disodium, and dexamethasone;
medium 3 contained tretinoin RA and SAG.
Preferably, the dosage of the bFGF is 10-20 ng/mL.
More preferably, bFGF is used in an amount of 10 ng/mL.
Preferably, the dosage of the EGF is 10-20 ng/mL.
More preferably, EGF is used in an amount of 10 ng/mL.
Preferably, the amount of the insulin-transferrin-selenium additive ITS is 1-2% by volume.
More preferably, the insulin-transferrin-selenium additive ITS is used in an amount of 1% by volume.
Preferably, the dosage of the tretinoin RA is 1-2 mu M.
More preferably, tretinoin RA is used in an amount of 1. mu.M.
Preferably, the amount of SAG is 1-5 μ M.
More preferably, SAG is used in an amount of 1. mu.M.
Preferably, the dosage of the glial cell line-derived neurotrophic factor GDNF is 10-20 ng/mL.
More preferably, the amount of glial cell line-derived neurotrophic factor GDNF is 10 ng/mL.
Preferably, the dosage of the brain-derived neurotrophic factor BDNF is 10-20 ng/mL.
More preferably, the brain derived neurotrophic factor BDNF is used in an amount of 10 ng/mL.
Preferably, the dosage of the ciliary neurotrophic factor CNTF is 10-20 ng/mL.
More preferably, the ciliary neurotrophic factor CNTF is used in an amount of 10 ng/mL.
Preferably, the dosage of the insulin-like growth factor IGF-I is 10-20 ng/mL.
More preferably, insulin-like growth factor IGF-I is administered in an amount of 10 ng/mL.
Preferably, the dosage of the bone morphogenetic protein BMP-4 is 50-100 ng/mL.
More preferably, the bone morphogenetic protein BMP-4 is used in an amount of 50 ng/mL.
Preferably, the dosage of the transforming growth factor TGF beta 1 is 100-500 ng/mL.
More preferably, the transforming growth factor TGF beta 1 is used in an amount of 100 ng/mL.
Preferably, the amount of the L-ascorbic acid AA is 0.2-0.5 mM.
More preferably, the amount of L-ascorbic acid AA is 0.2 mM.
Preferably, the amount of dibutyl CAMP is 0.1-0.5. mu.M.
More preferably, the amount of dibutyl CAMP is 0.1 μ M.
Preferably, the dosage of the beta-disodium glycerophosphate is 10-50 mM.
More preferably, the amount of disodium beta-glycerophosphate is 10 mM.
Preferably, the dosage of dexamethasone is 100-500 nM.
More preferably, dexamethasone is used in an amount of 100 nM.
Preferably, one or more of the culture media 1 to 3,
the culture medium 1 is DME/F-12 culture medium containing bFGF and EGF and NeurobasalTMA mixture of culture media;
the culture medium 2 is DME/F-12 culture medium containing insulin-transferrin-selenium additive ITS, retinoic acid RA, SAG, glial cell line-derived neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, insulin-like growth factor IGF-I, bone morphogenetic protein BMP-4, transforming growth factor TGF beta 1, L-ascorbic acid AA, dibutyl CAMP, beta-glycerophosphate disodium and dexamethasone, and NeurobasalTMA culture medium;
the culture medium 3 is MesenCult containing retinoic acid RA and SAGTM-ACF chondrogenic differentiation medium.
More preferably, DME/F-12 medium and NeurobasalTMThe volume ratio of the using amount of the culture medium is 0.9-1.1: 1.1 to 0.9.
Even more preferably, DME/F-12 medium and NeurobasalTMThe volume ratio of the using amount of the culture medium is 1: 1.
preferably, one or more of the culture media 1 to 3,
wherein the culture medium 1 contains N-2Supplement and B-27TMSupplement、GlutaMAXTMDME/F-12 medium and Neurobasal of beta-mercaptoethanol 2-mercaptoethanol, bFGF and EGFTMA mixture of culture media;
the culture medium 2 contains N-2Supplement and B-27TM Supplement、GlutaMAXTMBeta-mercaptoethanol 2-mercaptoethanol, insulin transferrin selenium supplement ITS, tretinoin RA, SAG, glial cell line-derived neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, insulin-like growth factor IGF-I, bone morphogenetic protein BMP-4, TGF beta 1, L-ascorbic acid AA, dibutyl CAMP, beta-glycerophosphate disodium, and dexamethasone DME/F-12 medium and NeurobasalTMA culture medium;
the culture medium 3 is MesenCult containing retinoic acid RA and SAGTM-ACF chondrogenic differentiation medium.
More preferably, the amount of N-2Supplement is 0.5 to 1 by volume.
Even more preferably, the amount of N-2Supplement is 1% by volume.
More preferably, B-27TMThe dosage of the Supplement is 1-2% by volume.
Even more preferably, B-27TMThe amount of Supplement used was 2% by volume.
More preferably, GlutaMAXTMThe amount of the surfactant is 0.5-1% by volume.
Even more preferably, GlutaMAXTMThe amount of (B) is 1% by volume.
More preferably, the amount of beta-mercaptoethanol 2-mercaptoethanol is 0.1 to 0.2 by volume.
Even more preferably, the amount of beta-mercaptoethanol 2-mercaptoethanol is 0.2% by volume.
The invention also claims a method for culturing spinal-spinal cord organoids, which uses any of the culture media to culture the NMPs.
Preferably, the method comprises the following steps,
s1, cleaning neuromesoderm progenitor cells NMPs, and digesting by using Accutase;
s2, resuspending the cells by using the culture medium 1, culturing,
s3, after the cells naturally agglomerate, transferring the cells into an ultra-low adsorption cell culture dish, and continuously culturing for 4-7 days by using the culture medium 1;
s4, culturing for 10-20 days by using the culture medium 2;
and S5, culturing for 20-50 days by using the culture medium 3.
More preferably, in step S3, Y27632 is further added to the first day medium of the culture.
More preferably, in step S3, 5 to 20 μ M Y27632 is added to the first day medium.
Still further preferably, in step S3, 10. mu. M Y27632 is added to the first day medium of the cultivation.
More preferably, in step S3, the culture is performed for 6 days.
More preferably, in step S4, the culture is performed for 12 days.
More preferably, in step S5, the culture is performed for 30 days.
More preferably, the neuromesodermal progenitor NMPs are induced by pluripotent stem cells PSCs.
Wherein the pluripotent stem cells PSCs are embryonic stem cell ESCs or induced pluripotent stem cell iPSCs, and include but are not limited to those obtained commercially, induced or prepared by other methods. The neural mesoderm progenitor NMPs obtained by inducing the pluripotent stem cells PSCs from various sources can be cultured according to the culture method to obtain the spinal-spinal cord organoid.
Even more preferably, the method for inducing the neuromesodermal progenitor NMPs by the pluripotent stem cell PSCs comprises the following steps: digesting pluripotent stem cells PSCs with the fusion degree of 75-85% by using Accutase; resuspending the cells using a medium comprising Y27632; culturing in a culture container treated with Matrigel in a culture medium containing Y27632 for 18-24 h, and culturing for 36-48 days in a neuromesoderm induction medium.
Still more preferably, pluripotent stem cell PSCs with a confluency of 80% are digested with Accutase.
Still more preferably, the culture vessel treated with Matrigel is cultured in a medium containing Y27632 for 24 hours and then cultured in a neuromesoderm-inducing medium for 2 days.
Still further preferably, the medium comprising Y27632 is mTeSR1 medium containing 5-20 μ MY 27632.
Still further preferably, the medium comprising Y27632 is mTeSR1 medium containing 10 μ M Y27632.
The invention also claims the application of any culture medium in the culture of spine-spinal cord organoids.
And the spine-spinal cord organoids cultured by the culture method also belong to the protection scope of the invention.
And, the use of the spine-spinal cord organoids as models of early development of the spine and/or spinal cord.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes neuromesoderm progenitor cells to induce and obtain the spine-spinal cord organoid, not only provides a complete in vitro model for the early development of the spine and the spinal cord, but also provides a theoretical basis for clarifying the normal development mechanism of the spine and the spinal cord and exploring the pathogenesis, treatment target and prevention and treatment means of related diseases.
Drawings
Fig. 1 is a flow chart of in vitro differentiation of NMPs into spine-spinal cord organoids (a) in example 4, a white light map of organoids at each stage during induced differentiation (B) (tissue differentiated for 3 days: bar 25 μm; tissue differentiated for 8 days: bar 25 μm; tissue differentiated for 20 days: bar 50 μm; tissue differentiated for 50 days: bar 250 μm) and a statistical map of organoids volume change during induced differentiation (C).
FIG. 2 shows the immunofluorescence staining in example 4 to detect the expression of different germ layer markers in early spine-spinal cord organoids (A) (upper row: bar 50 μm; lower row: bar 40 μm), and qPCR to detect the expression of different germ layer markers in early spine-spinal cord organoids (B).
FIG. 3 shows the results of immunofluorescence staining in example 4 for the detection of expression of different germ layer markers in mid-term spine-spinal cord organoids (A) (upper row: bar 50 μm; lower row: bar 70 μm), and qPCR for the detection of expression of different germ layer markers in mid-term spine-spinal cord organoids (B).
FIG. 4 shows the immunofluorescence staining assay of mature spine-spinal cord organoid expression (A) (bar 150 μm), hematoxylin-eosin and toluidine blue staining assay of late spine-spinal cord organoid tissue distribution (B) (left and middle bar 200 μm and right bar 70 μm), and qPCR assay of mid-term spine-spinal cord organoid expression of different germ layer markers (C).
Fig. 5 is a graph of the specific neuronal analysis (a) (bar 20 μm) in the late spine-spinal cord organoids, the calcium ion signal characteristics (B) (bar 100 μm) of the late spine-spinal cord organoids, the sodium potassium signal characteristics (C) and the action potential characteristics (D) of neurons in the mature spine-spinal cord organoids of example 5.
Fig. 6 is a diagram of kidney capsule transplantation pattern (a) of a spine-spinal cord organoid mouse in example 6, wherein kidney tissue (B) after transplantation of the organoid is harvested (bar 1cm), hematoxylin-eosin and toluidine blue staining is used for detecting the distribution of the region inside the tissue block (C) (upper row: bar 200 μm; lower row: bar 200 μm), and immunofluorescence staining is used for identifying the distribution characteristics of the region inside the tissue block (D) (first row: bar 150 μm; second row: bar 50 μm).
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The information of the materials and reagents used is shown in the following table:
Figure BDA0003362747160000071
EXAMPLE 1 spine-spinal cord organoid differentiation Medium A (for early differentiation)
A, make up
Contains N-2Supplement (1%, v/v), B-27TMSupplement(2％，v/v)、GlutaMAXTM(1%, v/v), β -mercaptoethanol 2-mercaptoethanol (0.2%, v/v), bFGF (10ng/mL), and EGF (10ng/mL) in a volume ratio of 1: 1 DME/F-12 medium and NeurobasalTMAnd (4) a culture medium.
Second, preparation method
(1) Preparing stock solutions of basic fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF), wherein the concentrations of the stock solutions are respectively 500 mu g/mL (bFGF) and 500 mu g/mL (EGF), and storing at-20 ℃.
(2) DME/F-12 medium and NeurobasalTMThe culture medium is mixed according to the volume ratio of 1: 1 mixing, adding N-2Supplement (100X), B-27TM Supplement(50×)、GlutaMAXTM(100X), beta-mercaptoethanol 2-mercaptoethanol (500X), bFGF (50000X, final concentration 10ng/mL), EGF (50000X, final concentration 10ng/mL), mixing, storing at 4 ℃, and using up within two weeks.
EXAMPLE 2 spine-spinal cord organoid differentiation Medium B (for metaphase of differentiation)
A, make up
Contains N-2Supplement (1%, v/v), B-27TMSupplement(2％，v/v)、GlutaMAXTM(1%, v/v), β -mercaptoethanol 2-mercaptoethanol (0.2%, v/v), insulin-transferrin-selenium supplement ITS (1%, v/v), retinoic acid RA (1 μ M), SAG (1 μ M), glial cell line-derived neurotrophic factor GDNF (10ng/mL), brain-derived neurotrophic factor BDNF (10ng/mL), ciliary neurotrophic factor CNTF (10ng/mL), insulin-like growth factor IGF-I (10ng/mL), bone morphogenetic protein BMP-4(50ng/mL), transforming growth factor TGF β 1(100ng/mL), L-ascorbic acid AA (0.2mM), Dibutyryl CAMP (0.1 μ M), β -glycerophosphate disodium (10mM), and dexamethasone (100nM) in a volume ratio of 1: 1 DME/F-12 medium and NeurobasalTMAnd (4) a culture medium.
Second, preparation method
(1) Preparing retinoic acid RA (stock solution concentration of 1mM), SHH channel agonist SAG (stock solution concentration of 10mM), glial cell line-derived neurotrophic factor GDNF (stock solution concentration of 100 mu g/mL), brain-derived neurotrophic factor BDNF (stock solution concentration of 100 mu g/mL), ciliary neurotrophic factor CNTF (stock solution concentration of 100 mu g/mL), insulin-like growth factor IGF-1 (stock solution concentration of 500 mu g/mL), bone morphogenetic protein BMP-4 (stock solution concentration 100 mug/mL), transforming growth factor TGF beta 1 (stock solution concentration 100 mug/mL), L-ascorbic acid AA (stock solution concentration 0.2M), adenosine dibutyrate cyclic phosphate Db-cAMP (stock solution concentration 0.5mM), beta-disodium glycerophosphate (stock solution concentration 1M), dexamethasone (stock solution concentration 5 mM).
(2) DME/F-12 medium and NeurobasalTMThe culture medium is mixed according to the volume ratio of 1: 1 mixing, adding N-2Supplement (100X), B-27TMSupplement(50×)、GlutaMAXTM(100X), beta-mercaptoethanol 2-mercaptoethanol (500. beta.0), insulin-transferrin-selenium supplement ITS (100. beta.2), retinoic acid RA (1000. beta.3, final concentration of 1. mu.M), SAG (10000. beta.4, final concentration of 1. mu.M), glial cell line-derived neurotrophic factor GDNF (10000. beta.6, final concentration of 10ng/mL), brain-derived neurotrophic factor BDNF (10000. beta.7, final concentration of 10ng/mL), ciliary neurotrophic factor CNTF (10000. beta.10, final concentration of 10ng/mL), IGF-1 (50000. beta.10 ng/mL), BMP4 (2000. beta.50 ng/mL), TGF. beta.11 (1000. beta.100 ng/mL), AA (1000. beta.2 mM), Db-5000. beta.5000. beta.1, final concentration of 0.1. mu.M), beta.5-glycerophosphate disodium (100. beta.5, cAMP, 100. beta.4, final concentration of 1. mu.M), and combinations of these compounds, 10mM final concentration), dexamethasone (50000X, 100nM final concentration), mixing, storing at 4 deg.C, and using up within two weeks.
EXAMPLE 3 spinal-spinal organoid differentiation Medium C (for late differentiation)
A, make up
MesenCult containing tretinoin RA (1. mu.M), and SAG (1. mu.M)TM-ACF chondrogenic differentiation medium.
Second, preparation method
(1) Retinoic acid RA (stock solution concentration 1mM), SHH pathway agonist SAG (stock solution concentration 10mM) were prepared and stored at-20 ℃.
(2) In MesenCultTMOn the basis of the culture medium for differentiating the cartilage from ACF, adding retinoic acid RA (1000X, final concentration of 1. mu.M) and SAG (10000X, final concentration of 1. mu.M), mixing, storing at 4 ℃ and using up within two weeks.
Example 4 method for differentiating NMPs into spinal-spinal organoids
First, experimental scheme
1. Method for differentiating NMPs into spinal-spinal cord organoids
(1) Inducing and differentiating the pluripotent stem cells PSCs (Day 0) to obtain neuromesodermal progenitor NMPs (Day 2);
preparing before induction: PSCs with the fusion degree of clone growth being about 80%; a cell culture dish coated with Matrigel (coating method: standing at 37 ℃ for at least 1 hour or more; standing overnight at 4 ℃).
② the PSCs culture medium is aspirated and washed twice by adding 1 XPBS.
③ removing 1 XPBS by aspiration, adding Accutase (about 0.05 mL/cm) to the well plate2) Digesting for 5-8 min at 37 ℃. The digestion solution was diluted with 5 volumes of 1 × PBS and mixed by pipetting. All liquid in the well plate was transferred to a centrifuge tube and centrifuged at 1100rpm for 4 min.
And fourthly, discarding the supernatant, resuspending the cell pellet by using 1-2 mL of mTeSR1 medium (containing 10 mu M Y27632), and then counting the cells.
Fifthly, absorbing the new culture dish by using a new culture dish coated by Matrigel, adding a proper amount of mTeSR Plus culture medium (about 0.2 mL/cm) containing 10 mu M Y276322) Wherein, the method for coating the new culture dish by the Matrigel comprises the following steps: the Matrigel was discarded after adding new medium overnight.
Sixthly, diluting the suspended cell suspension according to proper density (3.5 multiplied by 10)4cells/cm2) Dropping cell suspension into the culture dish, and gently shaking in a cross shape to uniformly distribute the cells in the culture dish. Placing into an incubator (37 ℃, 5% CO)2) Culturing in medium.
Seventhly, sucking out mTeSR1 culture medium containing 10 mu M Y27632 after culturing cells for 24 hours, adding the neuromesoderm induction culture medium, and culturing for about 2 days, wherein liquid is changed every day during the culture period, and then the NMPs are obtained.
(3) Obtaining spine-spinal cord organoids by PSCs-NMPs
Preparing before induction: NMPs obtained in step (1).
② the NMPs culture medium is aspirated and washed twice by adding 1 XPBS.
③ removing 1 XPBS by aspiration, adding Accutase (about 0.05 mL/cm) to the well plate2) And digesting at normal temperature for 1 min. The digestion solution was diluted with 5 volumes of 1 × PBS and mixed by pipetting. All liquid in the well plate was transferred to a centrifuge tube and centrifuged at 1100rpm for 4 min.
Fourthly, the supernatant is discarded, and 1 to 2mL of the spinal-spinal cord device of the embodiment 1 is usedAfter resuspending the cell pellet in organodifferentiation medium a (containing 10 μ M Y27632), the ratio of 1: 3 (e.g., six well plates, 1 six well cells to 3 six wells or 1T 25 flask) the cell suspension is dropped into the dish and the medium is supplemented to the appropriate volume (six wells to about 2mL, T25 to about 6mL) and gently shaken in a "cross" fashion to distribute the cells evenly in the dish. Placing into an incubator (37 ℃, 5% CO)2) Culturing in medium.
Fifthly, after one Day of culture, the cells are naturally rolled into clusters, the clusters of cells and the culture medium are transferred into an ultra-low adsorption cell culture dish and placed on a shaking table to be cultured at the rotating speed of 80rpm, and the culture medium (spinal-spinal cord organoid differentiation medium A of example 1, which does not contain 10 mu M Y27632) is replaced by half of liquid change every two days for 6 days (Day3-Day 8).
Sixthly, sucking all cell tissues in a centrifuge tube, naturally settling, sucking a supernatant, replacing the culture medium with the spinal-spinal cord organoid differentiation medium B in the example 2, culturing for about 12 days, and replacing the culture medium with half liquid every two days (Day9-Day 20).
And seventhly, sucking all cell tissues in a centrifuge tube, naturally settling, sucking a supernatant, replacing the spinal-spinal cord organoid differentiation medium C in the embodiment 3, continuously culturing for about 30 days, and half replacing the medium every two days (Day21-Day50) to obtain mature organoid tissues (spinal-spinal cord compound organoids) containing chondrocytes, osteoblasts and mature neurons.
2. Identification of spine-spinal cord organoids
The spinal cord develops through the differentiation process of neural stem cells, immature neurons, mature neurons, while the intraspinal skeleton is formed in a manner of endochondral osteogenesis, following the developmental pathway from mesodermal cells, chondrocytes, osteocytes. Therefore, the induced differentiation process is divided into three stages, and the expression of neural and cartilage/bone markers is detected separately at each stage.
(1) Early stage of differentiation (differentiation to Day8)
And identifying the organoid tissues differentiated for 8 days by immunofluorescence staining, and observing whether the inside of the organoid tissues has neural stem cells and mesenchymal stem cells at the same time. And whether both types of cells fit into a certain spatial orientation, i.e. whether mesenchymal stem cells are distributed closer to the outside of the tissue, and whether neural stem cells are distributed closer to the inside of the tissue.
The markers of the NMPs, the neural stem cells and the mesenchymal stem cells are identified by qPCR, and whether the expression of the markers of the NMPs is reduced and whether the expression of the markers of the neural stem cells and the mesenchymal stem cells is increased in the differentiation process are researched.
(2) Metaphase of differentiation (differentiation to Day20)
Organoid tissues differentiated for 20 days were identified by immunofluorescence staining to see if chondroprogenitor and immature neurons were present in the interior. Meanwhile, whether the cells of two germ layers have certain spatial arrangement or not forms a certain structure or not.
The expression of markers of stem/progenitor cells during differentiation was determined by qPCR to be decreased, while the expression of markers of specific progenitor cells (e.g., chondroprogenitor, motor neuron precursor cells) was increased.
(3) Late stage of differentiation (differentiation to Day50)
Organoid tissues differentiated for 50 days were identified by immunofluorescence staining to see if they had chondrocytes, osteoblasts and mature neurons inside. And whether the cells of two germ layers have certain spatial arrangement or not.
Cell types in organoid tissues were judged by paraffin sectioning and hematoxylin-eosin staining and toluidine blue staining.
The markers of more mature cells (chondrocytes, osteoblasts, mature neurons) and the expression of markers representing different segments of the trunk during differentiation were identified by qPCR for the increase in expression.
Second, experimental results
During the differentiation process, the cells formed spherical structures and gradually increased, with a diameter of about 150 μm for 8 days of differentiation, about 700 μm for 20 days of differentiation, and about 1.25mm for 50 days of differentiation (FIG. 1).
Cryo-sectioning and immunofluorescent staining of organoid tissues differentiated for 8 days (Day 8) revealed SOX2+ and NESTIN + cells representing neural stem cells (FIG. 2), as well as PDGFRA + and PDGFRB + cells representing mesenchymal stem cells (FIG. 2), and a population of intermediate neuronal progenitors (FIG. 2) with PAX2 +. From a spatial orientation, PDGFRA + and PDGFRB + cells were distributed closer to the outside of the tissue, while SOX2+, NESTIN + and PAX2+ cells were distributed closer to the inside of the tissue (fig. 2). The results of qPCR also showed similar gene expression profiles. In organoids in the early stage of differentiation, the expression of mRNA levels such as marker T, TBX6 of NMPs was decreased, while the expression levels of neural stem cell and mesenchymal stem cell markers such as PAX6, CDH6, MEOX2, TWIST1 were increased, using the NMPs stage (Day 2) as a control (fig. 2). This indicates that NMPs gradually differentiate towards cells of the downstream neuroand mesodermal lineage.
Cryo-sectioning and immunofluorescent staining of organoid tissues differentiated for 20 days (Day 20) revealed that the structure of the cell globules was further specialized, with SOX9+ chondroprogenitors appearing outside (fig. 3), which were specialized into "luminal" like structures, and immature neurons of TUJ1+ and DCX + appeared inside (fig. 3), while a portion of FN + mesodermal cells was also detected (fig. 3), indicating that the development of the cell globules was somewhat spatio-temporal specific. The results of qPCR showed that, in organoid tissues of early differentiation stage (Day 8) as control, the intermediate neuron progenitor markers were significantly down-regulated, the expression levels of early neural stem cell markers such as SOX2, NES, NKX3-2 were substantially maintained or increased, the expression levels of early mesenchymal stem cell markers such as TWIST1, MEOX2, etc. were decreased (fig. 3), the expression levels of early neuronal markers such as TUBB3, ASCL1, NEUROD1, DCX, ISL-1, etc. were significantly increased (fig. 3), and the expression levels of chondroprogenitor markers such as SOX9, MESP2, ACAN, IBSP, etc. were also significantly up-regulated (fig. 3). The expression of the key transcription factor RUNX2 of osteoblasts was not significantly changed, suggesting that there was no significant increase in osteogenic activity at this stage, which may be consistent with the characteristics of intrachondrogenic bone in the spine. The above results indicate that the inside of the cell tissue is continuously matured and further specialization of the cells occurs.
Frozen sections and immunofluorescence staining tests of organoid tissues differentiated for 50 days (Day 50) show that the organoid tissues are further specialized and mature in structure, chondrocytes of CTSB + and COL-2+ are shown on the outer side of the ball, and TUJ1+ and MAP2+ neurons surround the inner part of the ball (figure 3), and the structure is similar to the spine/spinal cord structure formed by the bone tissues of the somite surrounding the neurons in the in-vivo development process. Meanwhile, paraffin sections and hematoxylin-eosin staining and toluidine blue staining were performed to find that the structural characteristics of the cell balls were similar to those of immunofluorescence staining, and the cartilage tissue areas on the outer side were more deeply stained and the nerve tissue areas on the inner side were less stained (fig. 4). Partial regional toluidine blue staining in this area showed a "cartilage crater" characteristic (fig. 4), indicating the formation of mature chondrocytes in the organoid. The qPCR results showed that, in organoids in the late stage of differentiation (Day 50), mRNA levels of the neural progenitor cell marker ISL-1 were slightly decreased, while expression levels of early neuronal markers DCX, TUBB3, ASCL1, etc. were significantly increased and expression of MAP2 was maintained substantially unchanged, using organoid tissues in Day20 in the middle stage of differentiation as a control (figure 4). Meanwhile, the motor neuron marker HB9, the interneuron marker LBX1, and HOXC8, HOXB9 and the like representing different somite sites were significantly up-regulated in expression level on average (fig. 4). On the other hand, the expression levels of COL-2, CTSB, ACAN, IBSP, and the osteoblast markers ALP, RUNX2, COL-1, and OPN were also increased to some extent (FIG. 4).
Example 5 identification of functional neurons in spine-spinal cord organoids
First, experimental scheme
In addition to the spatial structural similarity, organoids should have similar physiological functions to tissues and organs in vivo, and therefore the differentiated mature organoid tissues obtained in example 4 were examined in relation thereto. And judging whether mature functional neurons exist in the organoid tissues according to the detection result.
Specifically, the method comprises the following steps: identifying whether motor neurons of HB9+ and interneurons represented by LHX1+ exist in the interior of the neuron by immunofluorescence staining; judging the maturity of the neuron by further detecting the electric signal; analyzing whether the organoids have neurons capable of generating calcium channel activity or not by using the Flou-4 calcium fluorescent probe dye for the calcium channel of the organoids; the ion channel signal and the action potential signal are detected by utilizing the patch clamp technology to judge whether the signals of the sodium ion inflow and the potassium ion outflow and the action potential signal generated along with the signals can be detected in the organoid.
Second, experimental results
The results of the calcium signal analysis of the cells by using the fluorescent probe dye of Flou-4 calcium show that a plurality of neurons which can continuously release calcium signals can be detected in the organoid tissues obtained in example 4 (FIG. 5), and the change of the flashing brightness of the neurons is converted into digital signals, so that a series of peak graphs (FIG. 5) which indicate the calcium signal intensity can be seen. Meanwhile, the examination of the ion channel signal and the electric signal of the organoid tissue obtained in example 4 by the patch clamp technique revealed that the signal of the sodium ion influx and the potassium ion efflux (fig. 5) and the action potential signal (fig. 5) generated thereby can be detected in the organoid tissue obtained in example 4. The above results further indicate that the organoid tissue induced to differentiate in example 4 has electrophysiological activity.
Example 6 verification of spinal-spinal organoid differentiation Capacity in vivo
First, experiment method
Organoid tissues of example 3 induced in vitro differentiation for about 30 days were transplanted into kidney envelopes of NCG mice of about 6 weeks of age, and whether tissues grew at the transplanted sites was observed after 1 to 3 months. The tissue mass was subjected to immunofluorescence and tissue stain staining to identify whether the tissue contains multiple cell types (nerve tissue, cartilage/bone tissue) simultaneously. Meanwhile, whether the cells in the transplanted tissue are human nucleic + cells is identified by immunofluorescence staining, so that whether the tissue block is derived from a human cell source is judged. Based on the above results, whether the organoid can differentiate and mature in the in vivo environment is analyzed.
Second, experimental results
Organoid tissues of example 3 induced in vitro differentiation for about 30 days were transplanted into kidney envelopes of NCG mice of about 6 weeks of age, and the tissues were removed after 1 to 3 months (FIG. 6), whereby it was found that white tissue masses having a certain hardness appeared in the transplanted sites (FIG. 6). Paraffin sections and hematoxylin-eosin, toluidine blue and safranin-fast green staining were performed on the tissues, and various tissue types were found in the tissues. As a result of toluidine blue staining, chondrocytes were found in the areas with purple-red color, and nerve cells were found in the areas with blue color (fig. 6). In the results of safranin-fast green staining, the region in orange-red color was chondrocytes (fig. 6). Meanwhile, the tissue formed by the transplanted cells is found to be human nucleic + cells by immunofluorescence staining (figure 6), which shows that the tissue block is derived from the transplanted human cell source and can be distinguished from the tissue of the mouse. Inside the tissue mass, the chondrocyte region of ACAN + and the mature neuron region of MAP2+ were detected (fig. 6), and a spatial structural feature was exhibited in which the cartilage region surrounds the nerve region. The results show that the organoids in the middle of differentiation can further develop and mature in an in vivo environment.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A culture medium for spinal-spinal organoids, comprising one or more of culture media 1 to 3,
wherein, the culture medium 1 contains bFGF and EGF;
the culture medium 2 contains insulin-transferrin-selenium additive ITS, retinoic acid RA, SAG, glial cell line-derived neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, insulin-like growth factor IGF-I, bone morphogenetic protein BMP-4, transforming growth factor TGF beta 1, L-ascorbic acid AA, Dibutyryl CAMP, beta-glycerol disodium phosphate and dexamethasone;
medium 3 contained tretinoin RA and SAG.
2. The culture medium of claim 1, comprising one or more of culture media 1 to 3,
the culture medium 1 is DME/F-12 culture medium containing bFGF and EGF and NeurobasalTMA mixture of culture media;
the culture medium 2 is DME/F-12 culture medium containing insulin-transferrin-selenium additive ITS, retinoic acid RA, SAG, glial cell line-derived neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, insulin-like growth factor IGF-I, bone morphogenetic protein BMP-4, transforming growth factor TGF beta 1, L-ascorbic acid AA, dibutyl CAMP, beta-glycerophosphate disodium and dexamethasone, and NeurobasalTMA culture medium;
the culture medium 3 is MesenCult containing retinoic acid RA and SAGTM-ACF chondrogenic differentiation medium.
3. The culture medium of claim 2, comprising one or more of culture media 1 to 3,
wherein the culture medium 1 contains N-2Supplement and B-27TMSupplement、GlutaMAXTMDME/F-12 medium and Neurobasal of beta-mercaptoethanol 2-mercaptoethanol, bFGF and EGFTMA mixture of culture media;
the culture medium 2 contains N-2Supplement and B-27TM Supplement、GlutaMAXTMBeta-mercaptoethanol 2-mercaptoethanol, insulin transferrin selenium supplement ITS, tretinoin RA, SAG, glial cell line-derived neurotrophic factor GDNF, brain-derived neurotrophic factor BDNF, ciliary neurotrophic factor CNTF, insulin-like growth factor IGF-I, bone morphogenetic protein BMP-4, TGF beta 1, L-ascorbic acid AA, dibutyl CAMP, beta-glycerophosphate disodium, and dexamethasone DME/F-12 medium and NeurobasalTMA culture medium;
the culture medium 3 is MesenCult containing retinoic acid RA and SAGTM-ACF chondrogenic differentiation medium.
4. A method for culturing spine-spinal cord organoids, wherein the medium according to any one of claims 1 to 3 is used to culture mesodermal progenitor NMPs.
5. The culture method according to claim 4, comprising the step of,
s1, cleaning neuromesoderm progenitor cells NMPs, and digesting by using Accutase;
s2. resuspending the cells with the medium 1 according to any one of claims 1 to 3 and culturing,
s3, after the cells naturally agglomerate, transferring the cells into an ultra-low adsorption cell culture dish, and continuously culturing for 4-6 days by using the culture medium 1 of any one of claims 1-3;
s4, culturing for 10-20 days by using the culture medium 2 as described in any one of claims 1-3;
s5, culturing the strain for 20 to 50 days by using the culture medium 3 as claimed in any one of claims 1 to 3.
6. The culture method according to claim 5, wherein the NMPs are induced by pluripotent stem cell PSCs.
7. The culture method according to claim 5, wherein the method for inducing the neuromesodermal progenitor NMPs by the pluripotent stem cells PSCs comprises: digesting pluripotent stem cells PSCs with the fusion degree of 75-85% by using Accutase; resuspending the cells using a medium comprising Y27632; culturing the culture container treated with Matrigel in a culture medium containing Y27632 for 18-24 hours, and then culturing the culture container in a neuromesoderm induction medium for 36-48 hours.
8. Use of a culture medium according to any one of claims 1 to 3 for culturing spine-spinal cord organoids.
9. The spine-spinal cord organoid cultured by the culturing method according to claim 8.
10. Use of the spine-spinal cord organoid of claim 9 as a model for early development of the spine and/or spinal cord.
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