Culture medium and culture method of hiPSC-sourced thyroid organoid

Abstract

The invention provides a culture medium and a culture method of a hiPSC-derived thyroid organ, and belongs to the technical field of thyroid organ culture. The invention relates to a culture method of a hiPSC-derived thyroid organ. The culture method comprises the following steps: (1) putting a resuscitated hiPSC-derived thyroid organ into a proliferation culture medium for culturing; and (2) when the thyroid organoid is cultured in the step (1) until the size of the thyroid organoid is 120-150 microns, replacing the proliferation culture medium with a differential culture medium, and continuously culturing. Sodium iodide with a specific concentration is added during thyroid organoid culture, so that the secretion amount of in-vitro thyroxine T4 of the organoid reaches 5ng / ml and is far higher than that (0.023 ng / ml) in the prior art. The invention provides a culture medium and a culture method capable of obviously promoting the maturity of the thyroid organoid, and the culture medium and the culture method have a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of thyroid organoid culture technology, and more particularly to a culture medium and culture method for hiPSC-derived thyroid organoids. Background Technology

[0002] The existing culture medium formulation and method for hiPSC-derived thyroid organoids are as follows: The constructed hiPSC-derived thyroid organoids are cultured in modified Ham's F12 medium (Thermo Fisher Scientific, Waltham, MA), supplemented with 5% bovine serum (Sigma-Aldrich, St. Louis, Missouri) and seven hormones, including bovine TSH (1 U / L), insulin (10 mg / L), hydrocortisone (0.4 mg / L), human transferrin (5 mg / L), glycyl-L-histidine-L-lysine acetate (10 μg / L), somatostatin (10 μg / L), and NaI (10 μg / L). After 48 hours, the used culture supernatant is harvested using the Millipore MAP thyroid magnetic bead method (Cat#RTHYMAG-30 K) to measure T4, according to the manufacturer's protocol. Unless otherwise specified, all chemical reagents were purchased from Sigma-Aldrich. All cells were cultured in matrix gel-coated culture dishes.

[0003] However, the in vitro thyroxine (T4) secretion of thyroid organoids cultured using existing hiPSC-derived thyroid organoid culture systems is only 0.023 ng / ml (Ma R, Shi R, Morshed SA, Latif R, Davies TF. Derivation and 97% Purification of Human Thyroid Cells From Dermal Fibroblasts. Front Endocrinol (Lausanne). 2020 Jul 15;11:446.), and T4 hormone secretion cannot be detected in in vivo animal experiments, indicating that the thyroid organoids cultured using this system are not mature enough. Therefore, it is necessary to explore new hiPSC-derived thyroid organoid culture systems. Summary of the Invention

[0004] The purpose of this invention is to provide a culture medium and culture method for hiPSC-derived thyroid organoids.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a proliferation culture medium for hiPSC-derived thyroid organoids, using DMEM-F12 medium as the basal medium, comprising the following components at final concentrations: A83-01 400~600 nM, head protein 20~30 ng / ml, B27 supplement 0.4~0.6 V / V%, Primocin 80~120 μg / ml, R-vertebral protein 1 150~250 ng / ml, nicotinamide 8~12 mM, Wnt3a 150~250 ng / ml, epidermal growth factor 15~25 ng / ml, heparin sodium solution 0.4~0.6 V / V%, fibroblast growth factor-2 15~25 ng / ml, GlutaMAX solution 0.15~0.25 mM, and Codonopsis pilosula extract 50~100 μg / ml.

[0006] This invention provides a differentiation culture medium for hiPSC-derived thyroid organoids, using Ham's F-12 medium and IMDM medium as the basal medium, comprising the following components at final concentrations: 1×GlutaMAX solution, BSA Fraction V 0.04~0.07V / V%, 0.5×B27 supplement, N2 supplement 0.003~0.005V / V%, α-thioglycerol 40~60μg / ml, ascorbic acid 80~120μg / ml, Primocin 80~120μg / ml, recombinant human fibroblast growth factor-2 200~300ng / ml, heparin sodium solution 80~120ng / ml, thyroid-stimulating hormone 0.2~0.4mIU / ml, recombinant human insulin-like growth factor-1 40~60ng / ml, 1×insulin-transferrin-selenium additive, and sodium iodide 0.8~1.2μM.

[0007] Preferably, the volume ratio of Ham's F-12 medium to IMDM medium is 20~30:60~90.

[0008] This invention provides the application of the aforementioned proliferation medium and / or differentiation medium in the culture of hiPSC-derived thyroid organoids.

[0009] This invention provides a method for culturing hiPSC-derived thyroid organoids, comprising the following steps: (1) The revived hiPSC-derived thyroid organoids were cultured in the aforementioned proliferation medium; (2) When the size of the thyroid organoid is 120~150μm after step (1), the proliferation medium is replaced with the differentiation medium and the culture continues.

[0010] Preferably, the culture temperature in steps (1) and (2) is 36~38℃.

[0011] Preferably, the culture medium is changed every 1 to 3 days during the cultivation process described in steps (1) and (2).

[0012] Preferably, the culture time in step (1) is 15 to 25 days.

[0013] Preferably, the culturing time in step (2) is 6 to 10 days.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a proliferation culture medium for hiPSC-derived thyroid organoids. The added A83-01 (a TGF-β receptor kinase inhibitor) inhibits epithelial-mesenchymal transition, maintains 3D structure, and promotes proliferation. Head protein maintains progenitor cells in an undifferentiated proliferative state, inhibiting differentiation, thus allowing organoids to continuously grow and passage. B27 supplementation is a key supplement for serum-free culture, promoting cell survival and proliferation. Primocin prevents and controls microbial contamination such as bacteria and mycoplasma. R-vertebral protein 1 acts as a Wnt signaling enhancer. Nicotinamide inhibits differentiation and improves metabolism. Wnt3a is a key morphogenetic and proliferation signal. Epidermal growth factor promotes rapid expansion of organoid cell number and volume. Heparin sodium solution enhances and prolongs the biological activity of fibroblast growth factor-2 (GF-2). GF-2 maintains the specific function of thyroid follicular cells and regulates morphology. GlutaMAX solution provides glutamine. Codonopsis pilosula extract may have specific protective, maturation-promoting, or functional-enhancing effects.

[0015] This invention also provides a differentiation culture medium for hiPSC-derived thyroid organoids, wherein the added GlutaMAX solution provides glutamine; BSA Fraction Vitamin V, as a carrier protein / protectant, can reduce cellular stress; B27 supplement is a key supplement for serum-free culture, promoting cell survival and proliferation; N2 supplement is a serum-free culture additive that provides core components such as insulin and transferrin, supporting cell proliferation; α-thioglycerol and ascorbic acid, as antioxidants, reduce oxidative stress in the culture medium and protect cells; Primocin can prevent and control microbial contamination such as bacteria and mycoplasma; recombinant human fibroblast growth factor-2 is used to maintain the specific function of thyroid follicular cells and regulate their morphology; heparin sodium solution can enhance and prolong the biological activity of fibroblast growth factor-2; thyroid-stimulating hormone, as a key differentiation factor, mimics physiological signals in vivo and strongly drives the maturation and hormone synthesis function of thyroid follicular cells; recombinant human insulin-like growth factor-1 provides survival / proliferation signals; insulin-transferrin-selenium additive is a core metabolic supplement: insulin promotes glucose metabolism, transferrin supplies iron, and selenium is a component of antioxidant enzymes; sodium iodide, as a raw material for hormone synthesis, provides the iodine element necessary for the synthesis of thyroid hormones (T3 / T4).

[0016] This invention involves adding a specific concentration of sodium iodide during the culture of thyroid organoids, resulting in an in vitro thyroxine (T4) secretion level of 5 ng / ml, significantly higher than existing technologies (0.023 ng / ml). This invention provides a culture medium and method that can significantly promote the maturation of thyroid organoids, and has broad application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The microstructure of the thyroid organoids proliferated and cultured in Example 1; Figure 2 The microstructure of the differentiated and cultured thyroid organoids in Example 1; Figure 3 This illustrates the effect of different concentrations of sodium iodide on the in vitro secretion of thyroxine T4 by thyroid organoids, as shown in Example 2. Figure 4 This illustrates the effect of different proliferation culture media on the fold increase in the volume of thyroid organoids in Example 3. Detailed Implementation

[0019] This invention provides a proliferation culture medium for hiPSC-derived thyroid organoids, using DMEM-F12 medium as the basal medium, comprising the following components at final concentrations: A83-01 400~600 nM, head protein 20~30 ng / ml, B27 supplement 0.4~0.6 V / V%, Primocin 80~120 μg / ml, R-vertebral protein 1 150~250 ng / ml, nicotinamide 8~12 mM, Wnt3a 150~250 ng / ml, epidermal growth factor 15~25 ng / ml, heparin sodium solution 0.4~0.6 V / V%, fibroblast growth factor-2 15~25 ng / ml, GlutaMAX solution 0.15~0.25 mM, and Codonopsis pilosula extract 50~100 μg / ml.

[0020] In this invention, the proliferation culture medium preferably comprises the following components at final concentrations: A83-01 450~550 nM, head protein 22~28 ng / ml, B27 supplement 0.45~0.55 V / V%, Primocin 90~110 μg / ml, R-vertebral protein 1 180~220 ng / ml, nicotinamide 9~11 mM, Wnt3a 180~220 ng / ml, epidermal growth factor 18~22 ng / ml, heparin sodium solution 0.45~0.55 V / V%, fibroblast growth factor-2 18~22 ng / ml, GlutaMAX solution 0.18~0.22 mM, and Codonopsis pilosula extract 65~85 μg / ml.

[0021] In this invention, the proliferation culture medium is further preferably composed of the following components at final concentrations: A83-01 500 nM, head protein 25 ng / ml, B27 supplement 0.5 V / V%, Primocin 100 μg / ml, R-vertebral protein 1 200 ng / ml, nicotinamide 10 mM, Wnt3a 200 ng / ml, epidermal growth factor 20 ng / ml, heparin sodium solution 0.5 V / V%, fibroblast growth factor-2 20 ng / ml, GlutaMAX solution 0.2 mM, and Codonopsis pilosula extract 75 μg / ml.

[0022] This invention provides a differentiation culture medium for hiPSC-derived thyroid organoids, using Ham's F-12 medium and IMDM medium as the basal medium, comprising the following components at final concentrations: 1×GlutaMAX solution, BSA Fraction V 0.04~0.07V / V%, 0.5×B27 supplement, N2 supplement 0.003~0.005V / V%, α-thioglycerol 40~60μg / ml, ascorbic acid 80~120μg / ml, Primocin 80~120μg / ml, recombinant human fibroblast growth factor-2 200~300ng / ml, heparin sodium solution 80~120ng / ml, thyroid-stimulating hormone 0.2~0.4mIU / ml, recombinant human insulin-like growth factor-1 40~60ng / ml, 1×insulin-transferrin-selenium additive, and sodium iodide 0.8~1.2μM.

[0023] In this invention, the differentiation culture medium preferably comprises the following components at final concentrations: 1×GlutaMAX solution, BSA Fraction V 0.05~0.06V / V%, 0.5×B27 supplement, N2 supplement 0.0035~0.0045V / V%, α-thioglycerol 45~55μg / ml, ascorbic acid 90~110μg / ml, Primocin 90~110μg / ml, recombinant human fibroblast growth factor-2 220~280ng / ml, heparin sodium solution 90~110ng / ml, thyroid-stimulating hormone 0.25~0.35mIU / ml, recombinant human insulin-like growth factor-1 45~55ng / ml, 1×insulin-transferrin-selenium additive, and sodium iodide 0.9~1.1μM.

[0024] In this invention, the differentiation culture medium is further preferably composed of the following components at final concentrations: 1×GlutaMAX solution, BSA Fraction V 0.05625V / V%, 0.5×B27 supplement, N2 supplement 0.004V / V%, α-thioglycerol 50μg / ml, ascorbic acid 100μg / ml, Primocin 100μg / ml, recombinant human fibroblast growth factor-2 250ng / ml, heparin sodium solution 100ng / ml, thyroid-stimulating hormone 0.3mIU / ml, recombinant human insulin-like growth factor-1 50ng / ml, 1×insulin-transferrin-selenium additive, and sodium iodide 1μM.

[0025] In this invention, the volume ratio of Ham's F-12 medium to IMDM medium is 20~30:60~90, preferably 22~28:65~85, more preferably 24~26:70~80, and even more preferably 25:75.

[0026] This invention provides the application of the aforementioned proliferation medium and / or differentiation medium in the culture of hiPSC-derived thyroid organoids.

[0027] This invention provides a method for culturing hiPSC-derived thyroid organoids, comprising the following steps: (1) The revived hiPSC-derived thyroid organoids were cultured in the aforementioned proliferation medium; (2) When the size of the thyroid organoid is 120~150μm after step (1), the proliferation medium is replaced with the differentiation medium and the culture continues.

[0028] In this invention, the cultivation temperature in steps (1) and (2) is 36~38℃, preferably 36.5~37.5℃, and more preferably 37℃.

[0029] In this invention, the culture medium is changed every 1 to 3 days during the cultivation process described in steps (1) and (2), preferably every 2 days.

[0030] In this invention, the culture time in step (1) is 15 to 25 days, preferably 18 to 22 days, and more preferably 20 days.

[0031] In this invention, the culturing time in step (2) is 6 to 10 days.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. The Codonopsis pilosula extract, Astragalus membranaceus extract, Astragalus polysaccharide, and Astragaloside A used in the following embodiments were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. The product code for Codonopsis pilosula extract is S28280-100g, the product code for Astragalus membranaceus extract is S27211-100g, the product code for Astragalus polysaccharide is S27818-1g, and the product code for Astragaloside A is V97949-5mg.

[0033] Example 1

[0034] 1. Preparation of culture medium

[0035] Prepare proliferation culture medium according to the components and dosages shown in Table 1, and prepare differentiation culture medium according to the components and dosages shown in Table 2.

[0036] Table 1. Components and dosage of proliferation culture medium

[0037] Table 2. Components and dosage of differentiation culture medium

[0038] 2. Culture methods for hiPSC-derived thyroid organoids

[0039] (1) Proliferation culture

[0040] Resuscitated hiPSC-derived thyroid organoids were cultured in a proliferation system, with 400 μl of proliferation medium added to each 48-well plate. The plates were incubated at 37°C in a CO2 incubator for 20 days, with the medium changed every 2 days. When the thyroid organoids measured to be 120–150 μm in size under a microscope, the culture medium was replaced. Figure 1 ).

[0041] (2) Differentiation culture

[0042] The proliferation medium was replaced with differentiation medium, and 600 μl of medium was added to each 48-well plate. The plates were incubated at 37°C in a CO2 incubator for 8 days, with the medium changed every 2 days. Under a microscope, the thyroid organoids were observed to be approximately 200 μm in size. Figure 2 Immunofluorescence results showed the presence of follicular structures.

[0043] Example 2

[0044] This example investigated the effect of sodium iodide addition and concentration in the differentiation medium on the in vitro thyroxine (T4) secretion from cultured thyroid organoids. Four treatment groups were set up: control (C, no sodium iodide added), 0.5 μM, 1 μM, and 5 μM. After culturing according to the method in Example 1, the T4 concentration in the supernatant was measured using a T4 ELISA kit. The specific detection method is as follows: 1. Preparation before testing (1) Take the kit out of the refrigerator 10 minutes in advance and allow it to equilibrate to room temperature.

[0045] (2) Preparation of standard gradient working solutions: Add 1 ml of universal diluent to the lyophilized standard, let stand for 15 minutes until it is completely dissolved, and then mix gently (concentration is 10 ng / ml). Then dilute according to the following concentrations: 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml, 0.312 ng / ml, 0.156 ng / ml, 0 ng / ml.

[0046] Dilute using a serial dilution method: Take 7 EP tubes, add 500µl of general diluent to each tube, and pipette 500µl of the 10ng / ml standard working solution into the first EP tube and mix well to prepare a 5ng / ml standard working solution. Repeat this process for the subsequent tubes. The last tube is used as a blank well; it is not necessary to pipette liquid from the second-to-last tube.

[0047] (3) Preparation of Biotin-antibody working solution: 15 minutes before use, centrifuge 100× concentrated Biotin-antibody at 1000×g for 1 minute, and dilute 100× concentrated Biotin-antibody to 1× working concentration with universal diluent.

[0048] (4) Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge 100× concentrated enzyme conjugate at 1000×g for 1 minute, and dilute 100× concentrated enzyme conjugate to 1× working concentration with general diluent.

[0049] (5) Preparation of 1× washing solution: Take 10ml of 20× washing solution into 190ml of distilled water.

[0050] 2. Operating Steps

[0051] (1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining strips in a self-sealing bag and put them back at 4℃.

[0052] (2) Sample addition: Add 50 µl of sample or standard of different concentrations to each well. Add 50 µl of universal diluent to each blank well, followed by 50 µl of Biotin antibody working solution to each well. Cover with sealing film and incubate at 37°C for 60 minutes.

[0053] (3) Washing the plate: Discard the liquid, add 300µl of 1× washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times.

[0054] (4) Add enzyme conjugate working solution: Add 100µl of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0055] (5) Washing the plate: Discard the liquid and wash the plate 5 times according to the method in step (3).

[0056] (6) Add substrate: Add 90µl of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes.

[0057] (7) Add stop solution: Take out the microplate, add 50µl of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450nm.

[0058] The results are as follows Figure 3 As shown, at a sodium iodide concentration of 1 μM, the concentration of T4 secreted by thyroid organoids can reach 5 ng / ml.

[0059] Example 3

[0060] This example investigated the effect of proliferation culture medium on the proliferation of thyroid organoids. The proliferation culture medium from Example 1 was used as the experimental group (75 μg / ml Codonopsis pilosula group), and the following control culture medium was set up: Bozhen Proliferation: A culture system for the proliferation of thyroid organoids, purchased from Bozhen Biotechnology Co., Ltd., catalog number: K2305-FTE; Self-developed system: Compared with the proliferation culture medium in Example 1, no Codonopsis pilosula extract was added; 75μg / ml Astragalus Group: Astragalus extract with a final concentration of 75μg / ml was added to the self-developed system. 75μg / ml Codonopsis and Astragalus self-developed system: Based on the self-developed system, a mixture of Codonopsis pilosula extract and Astragalus membranaceus extract was added, with a mass ratio of Codonopsis pilosula to Astragalus membranaceus of 1:1, and the final concentration of the mixture was 75μg / ml; Astragalus polysaccharide: Astragalus polysaccharide was added to the self-developed system to a final concentration of 75 μg / ml; Astragaloside A: Astragaloside A was added to the self-developed system to a final concentration of 75 μg / ml.

[0061] The revived hiPSC-derived thyroid organoids were placed in the above-mentioned culture medium and cultured according to the proliferation culture method described in Example 1. After 20 days of culture, the volume increase of the thyroid organoids in each treatment group was calculated. The results are as follows: Figure 4 As shown in the figure, the thyroid organoids obtained by culture in the proliferation medium in Example 1 showed the greatest increase in volume.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A proliferation culture medium for hiPSC-derived thyroid organoids, characterized in that, The basal medium was DMEM-F12, which included the following components at final concentrations: A83-01 400-600 nM, head protein 20-30 ng / ml, B27 supplement 0.4-0.6 V / V%, Primocin 80-120 μg / ml, R-vertebral protein 1 150-250 ng / ml, nicotinamide 8-12 mM, Wnt3a 150-250 ng / ml, epidermal growth factor 15-25 ng / ml, heparin sodium solution 0.4-0.6 V / V%, fibroblast growth factor-2 15-25 ng / ml, GlutaMAX solution 0.15-0.25 mM, and Codonopsis pilosula extract 50-100 μg / ml.

2. A differentiation culture medium for hiPSC-derived thyroid organoids, characterized in that, Ham's F-12 medium and IMDM medium were used as the basal medium, containing the following components at final concentrations: 1×GlutaMAX solution, BSA Fraction V 0.04~0.07V / V%, 0.5×B27 supplement, N2 supplement 0.003~0.005V / V%, α-thioglycerol 40~60μg / ml, ascorbic acid 80~120μg / ml, Primocin 80~120μg / ml, recombinant human fibroblast growth factor-2 200~300ng / ml, heparin sodium solution 80~120ng / ml, thyroid-stimulating hormone 0.2~0.4mIU / ml, recombinant human insulin-like growth factor-1 40~60ng / ml, 1×insulin-transferrin-selenium supplement, and sodium iodide 0.8~1.2μM.

3. The differentiation culture medium according to claim 2, characterized in that, The volume ratio of Ham's F-12 medium to the IMDM medium is 20~30:60~90.

4. The use of the proliferation medium of claim 1 and / or the differentiation medium of claim 2 or 3 in the culture of hiPSC-derived thyroid organoids.

5. A method for culturing hiPSC-derived thyroid organoids, characterized in that, Includes the following steps: (1) The revived hiPSC-derived thyroid organoids are cultured in the proliferation medium described in claim 1; (2) When the size of the thyroid organoid is 120~150μm after step (1), the proliferation medium is replaced with the differentiation medium described in claim 2 and the culture is continued.

6. The method according to claim 5, characterized in that, The cultivation temperature described in steps (1) and (2) is 36~38℃.

7. The method according to claim 5, characterized in that, The culture medium in steps (1) and (2) should be changed every 1 to 3 days.

8. The method according to claim 5, characterized in that, The culture time described in step (1) is 15 to 25 days.

9. The method according to claim 5, characterized in that, The culturing time described in step (2) is 6 to 10 days.

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