Culture medium for preparing human-derived Merkel cell model and construction method

Abstract

The invention discloses a culture medium for preparing a human-derived Merkel cell model and a construction method, and relates to the technical field of biologication.The key points of the technical scheme are that the culture medium comprises a culture medium E, and the culture medium E is formed by adding PRCi into an OMM culture medium; the OMM is prepared by adding GlutaMax (GlutaMax), B-27 (B-27) Minus, N2 (N2) suppliers, Penicilin-Streptomycin and 2-Mercaptoethanol into an Advanced DMEM (Dulbecco's Modified Eagle Medium) / F12 (Fullcap 12) culture medium and a Neurobasic culture medium, and the OMM is prepared by adding GlutaMax, B-27 Minus, N2 (N2) suppliers, Penicilin-Streptomycin and 2-Mercaptoethanol into a medium; and the PRCi comprises EED226, PRT4165 and GSK126 (glutamine synthase 126). According to the invention, a small molecule compound combination PRCi (EED226 + PRT4165 + GSK126) is added into a culture medium, so that the amount of MC in the organoid can be remarkably increased, and a human-derived system is established for researching the MC.

Description

[0001] TECHNICAL FIELD

[0002] The present application relates to the technical field of biotechnology, and more particularly, it relates to a culture medium for preparing a human Merkel cell model and a construction method. BACKGROUND

[0003] Skin is an important organ of the human body, not only protecting the body from external harm, but also helping the body to perceive the external environment, and touch is an important part of the perception function, in which fine touch is mediated by touch receptors Merkel cells (MC). Clinically, MC regeneration and touch repair after skin burns and large-area trauma are difficult points in treatment; skin itching and ectopic pain and other diseases are also directly related to the number and abnormal function of MC. However, due to the small number of MC in the skin, the in vitro culture system is not mature, the research is difficult, the related research develops slowly, and the research of human MC is especially lacking. The present project intends to establish a research model of human MC through a human pluripotent stem cell differentiation system, to provide a platform for the research of touch generation and touch-related diseases in clinical treatment, and to provide clues for the development of human touch receptors and artificial perception skin.

[0004] The prior art differentiates human pluripotent stem cells (hPSC) into skin organoids in the form of EB, and the Merkel cells (MC) in the organoids are less. In view of this, the present application provides a culture medium for preparing a human Merkel cell model. SUMMARY

[0005] The purpose of the present application is to provide a culture medium for preparing a human Merkel cell model and a construction method, which can produce more MC in the organoids.

[0006] The above technical purpose of the present application is achieved by the following technical scheme:

[0007] The present application provides a culture medium for preparing a human Merkel cell model in the first aspect, which comprises a culture medium E,

[0008] The culture medium E adds PRCi in the OMM culture medium;

[0009] The PRCi comprises EED226, PRT4165 and GSK126.

[0010] In combination with the first aspect, the present application is further provided as: further comprising culture medium A, culture medium B, culture medium C and culture medium D;

[0011] Culture medium A: E8-Flex culture medium;

[0012] Medium B: 2% Matrigel, 10 μΜ SB431542, 4 ng / ml basic-FGF and 10 ng / ml BMP-4 added to E6 medium;

[0013] Medium C: 1 μΜ LDN and 250 ng / ml bFGF added to E6 medium;

[0014] Medium D: 1% Matrigel added to OMM medium.

[0015] In combination with the first aspect, the application further provides that the concentrations of EED226, PRT4165 and GSK126 are 10 μΜ, 10 μΜ and 5 μΜ.

[0016] In combination with the first aspect, the application further provides that the OMM is GlutaMax, B-27 Minus, N2 supplements, Penicillin-Streptomycin (PS) and 2-Mercaptoethanol added to Advanced DMEM / F12 and Neurobasal medium.

[0017] The second aspect of the application provides a construction method for preparing a human Merkel cell model, characterized by using the above-mentioned medium, comprising the following steps:

[0018] D-2: Take the medium A + 10Y resuspended cells and add them to the adsorption plate containing medium A + 10Y, centrifuge and place in the incubator for culture;

[0019] D-1: Supplement fresh E8-Flex medium;

[0020] D-0: Take out the cell spheres, wash them, and transfer the EBs to a new adsorption plate containing medium B;

[0021] D3: Culture with medium C supplement;

[0022] D6: Add fresh E6 medium to supplement the liquid;

[0023] D8 and D10: Half liquid exchange, suck out the old medium and add fresh E6 medium;

[0024] D12: Transfer the cell spheres to a low-adsorption well plate containing medium D, and make the well plate vibrate on a vibrating screen;

[0025] D15: Half liquid exchange, add new medium D;

[0026] D18 to D42 stage: half medium exchange every three days, aspirate old medium, add OMM medium;

[0027] D42: add PRCi to the medium, i.e. use medium E for culture, continue to culture for 2-4 weeks.

[0028] In combination with the second aspect, the application is further provided that in D-2, the centrifugation is performed by using a centrifuge at 200g for 3min to make the cells aggregate.

[0029] In combination with the second aspect, the application is further provided that in D0, each well of the adsorption plate contains 100ul of medium B.

[0030] In combination with the second aspect, the application is further provided that in D3, the medium C in each well contains 25ul.

[0031] In combination with the second aspect, the application is further provided that in D6, each well contains 200ul of medium.

[0032] In combination with the second aspect, the application is further provided that in D8 and D10, half medium exchange is performed, i.e. aspirate 100ul of old medium and add 100ul of fresh E6 medium.

[0033] In combination with the second aspect, the application is further provided that the method for keeping the cell ball in a floating and good nutrient flow state is to oscillate the well plate on a vibration sieve.

[0034] In summary, the application has the following beneficial effects:

[0035] In the published protocol, the culture system using E8 medium and Vitronectin (VTN) coated medium (E8-VTN) is used, and in practice, we find that the stem cells cultured in this system grow slowly and unstably. In the present application, the medium mTeSR plus is used in combination with the culture system of Matrigel (354277, Corning) (mTeSR-M), and in this system, the stem cells grow well and stably. In the first day of the EB formation stage, 20ul of Y-27632 in the protocol is replaced by 10ul of Y-27632, and in the differentiation stage, the concentration of BMP is adjusted to 10ng / ml. In the later culture, the application can significantly increase the amount of MC in the organoids by adding small molecule compound combination PRCi (EED226+PRT4165+GSK126) to the medium, thereby establishing a human system for studying MC. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a schematic diagram of the induction process of the organoids of the application;

[0037] Figure 2 Figure 7 is a bar graph showing the number of K20+ MCs in the PRCi group of the present application (A) and whole tissue staining (B) (wherein the number of K20+ MCs in the PRCi group was significantly increased; **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0039] Main reagents

[0040] Reagent name Catalogue number Company Accutase A1110501 Gibco mTeSRplus 100-0276 Stemcell Y-27632 1254-1 / 10 Tocris Matrigel 354277 Corning Matrigel 354230 Corning E8-Flex A2858501 Gibco E6 A1516401 Gibco Advanced DMEM / F12 12634010 Gibco Neurobasal 21103049 Gibco N2 17502048 Gibco B27 minus 12587010 Gibco SB431542 04-0010-05 Stemgent bFGF 100-18B Peprotech BMP4 314-BP RD LDN 04-0074-02 Stemgent GlutaMax 35050061 Gibco 2-Mercaptoethanol 17502048 Gibco Penicillin-Streptomycin C3420 PI

[0041] Example 1:

[0042] Configuration of culture medium

[0043] E8-Flex culture medium was used to configure medium A for standby;

[0044] 2% Matrigel (354230, Corning), 10 μΜ SB431542, 4 ng / ml basic-FGF and 10 ng / ml BMP-4 were added to E6 (100 ul) culture medium to configure medium B for standby;

[0045] 25 ul of E6 base medium containing 1 μΜ LDN (Stemgent) + 250 ng / ml bFGF was added to medium B to obtain medium C;

[0046] 1% Matrigel (354230) was added to OMM to configure medium D for standby;

[0047] In this embodiment, the following method was used to prepare:

[0048] Advanced DMEM / F12 and Neurobasal medium 1:1 + 1x GlutaMax TM +0.5x B-27Minus+0.5x N2 supplements+1x Glutamax+0.1 mM 2-Mercaptoethanol (Gibco). OMM culture medium was configured for standby;

[0049] In this embodiment, take 100 ml of OMM medium as an example, 48.2 ml of Advanced DMEM / F12 + 48.2 ml of Neurobasal medium + 1 ml of B-27 Minus + 0.5 ml of N2 supplements + 1 ml of Glutamax + 100 ul of 2-Mercaptoethanol + 1 ml of PS.

[0050] Add PRci (10 mM EED + 10 mM PRT4165 + 5 mM GSK126) to OMM, and the medium E is obtained;

[0051] A method for constructing a human Merkel cell model, as shown in Figure 1 comprises the following steps:

[0052] hPSCs were maintained in mTeSR plus medium and passaged every 3-6 days;

[0053] The following are the specific differentiation steps of hPSC-derived SKO:

[0054] 1. Digestion, aspirate the medium, wash once with PBS, add Accutase and incubate at 37°C for 4 min, gently blow the cells and collect them in a 15 ml centrifuge tube, centrifuge at 400g for 5 min;

[0055] 2. Resuspend and count, resuspend hPSCs with E8-Flex medium + 10 mM Y-27632 (10Y) and count;

[0056] 3. EB formation, take a certain proportion of cells, 4000 cells / 100 ul system, culture in a low-adsorption U-shaped 96-well plate, add medium A (medium E8-Flex)

[0057] +10Y, to make the cells aggregate, centrifuge at 200g for 3 min, observe the cell aggregation into pieces, and then place in an incubator for culture. At this time, it is counted as D-2;

[0058] 4. Liquid replenishment, D-1, add fresh E8-Flex medium 100 ul;

[0059] 5. Induction begins D0, aspirate the cell ball, wash with E6, transfer the EB to a new U-shaped 96-well plate containing differentiation medium, which contains medium B, 100 ul of medium per well: E6 + 2% Matrigel (354230, Corning) + 10 mM SB431542

[0060] +4 ng / ml basic-FGF + 10 ng / ml BMP-4;

[0061] 6. Induction D3, add 25ul of E6 base medium containing 1 uM LDN (Stemgent) + 250ng / ml bFGF to the above medium, final system 125ul;

[0062] 7. D6, supplement, add 75ul of fresh E6 medium, total system 200ul / well;

[0063] 8. D8 and D10, half medium change, remove 100ul of old medium, add 100ul of fresh E6 medium;

[0064] 9. Induce epidermal self-assembly, D12, transfer all cell spheroids to low attachment 24-well plate single well, containing medium D (OMM + 1% Matrigel (354230)) medium, 500ul of medium per well, in order to keep the cell spheroids floating and good nutrition flow state, 24-well plate is shaken on a shaker at a speed of 65rpm;

[0065] 10. D15, half medium change, remove 250ul of old medium, add 300ul of fresh OMM + 1% Matrigel.

[0066] 11. Medium change, D18, from D18-D42, half medium change every three days, medium is OMM, according to the size of the organoid, the amount of medium can be increased and the medium change period can be shortened.

[0067] 12. D42, add PRCi (10uM EED + 10uM PRT4165 + 5uM GSK126) to the medium, i.e. medium E, continue to culture for 2-4 weeks.

[0068]

[0069] Comparative Example 1:

[0070] The control group continues to be cultured with OMM medium, and samples are collected at the same time.

[0071] The number of MCs in the organoids in Example 1 and Comparative Example 1 was detected by immunofluorescence staining of MC marker K20, and the results are shown in Figure 2 Example 1 (PRCi group) K20+ MCs increased significantly.

[0072] ​Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent ones. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A culture medium for preparing a human Merkel cell model, characterized in that: The culture medium includes culture medium E. The culture medium E is OMM medium supplemented with PRCi; The PRCi includes EED226, PRT4165, and GSK126.

2. The culture medium for preparing a human Merkel cell model according to claim 1, characterized in that: It also includes culture medium A, culture medium B, culture medium C, and culture medium D; Culture medium A: E8-Flex medium; Culture medium B: E6 medium supplemented with 2% Matrigel, 10 μM SB431542, 4 ng / ml basic-FGF and 10 ng / ml BMP-4; Culture medium C: Add 1 μM LDN and 250 ng / ml bFGF to E6 medium; Culture medium D: Add 1% Matrigel to OMM medium.

3. The culture medium for preparing a human Merkel cell model according to claim 1, characterized in that: The concentrations of EED226, PRT4165, and GSK126 were 10 μM, 10 μM, and 5 μM, respectively.

4. The culture medium for preparing a human Merkel cell model according to any one of claims 1-2, characterized in that: The OMM is prepared by adding GlutaMax, B-27Minus, N2supplements, Penicil lin-Streptomycin and 2-Mercaptoethanol to Advanced DMEM / F12 and Neurobasal media.

5. A method for constructing a human Merkel cell model, characterized in that: The culture medium according to claim 2 comprises the following steps: D-2: Resuspend the cells in culture medium A+10Y, add them to an adsorption plate containing culture medium A+10Y, centrifuge, and incubate in an incubator. D-1: Replenish with fresh E8-Flex medium; D-0: Aspirate the cell spheres, wash them, and transfer EB to a new adsorption plate containing culture medium B. D3: Cultured in culture medium C supplemented with D3; D6: Replenish fluid by adding fresh E6 culture medium; D8 and D10: Half of the medium was changed; the old medium was aspirated and fresh E6 medium was added. D12: Transfer cell spheres to low-adsorption well plates containing culture medium D, and then vibrate the well plates on a vibrating screen. D15: Half-change the medium, add fresh medium D; D18 to D42 stage: Change the medium every three and a half days, remove the old medium, and add OMM medium; D42: Add PRCi to the culture medium, i.e., use culture medium E, and continue culturing for 2-4 weeks.

6. The construction method according to claim 5, characterized in that: In D-2, the centrifugation was performed using a centrifuge at 200g for 3 minutes to induce cell aggregation.

7. The construction method according to claim 5, characterized in that: In D0, each well of the adsorption plate contains 100 μL of culture medium B.

8. The construction method according to claim 5, characterized in that: In D3, the C content of the culture medium in each well is 25 μL.

9. The construction method according to claim 5, characterized in that: In D6, each well contains 200 μL of culture medium.

10. The construction method according to claim 5, characterized in that: On days 8 and 10, the medium was partially changed by aspirating 100 μL of the old medium and adding 100 μL of fresh E6 medium.

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