A method for establishing a bactrian camel intestinal organoid model and application thereof

Abstract

The application provides a method for establishing a Bactrian camel intestinal tract organoid model and application thereof, and the method is as follows: mucous mucus of Bactrian camel intestinal tract tissue is scraped off, washed, digested, washed again, crypt cells are scraped, cells are suspended in a culture medium, screened, filtrate is collected, centrifuged, supernatant is discarded, cells are resuspended in a DMEM / F12 culture medium containing bovine serum albumin to obtain a cell suspension; matrix glue is added to the cell suspension, mixed uniformly, inoculated, grown, grown culture medium is added, and then the Bactrian camel intestinal tract organoid model is obtained; and the obtained Bactrian camel intestinal tract organoid model is subcultured. The application also provides application of the Bactrian camel intestinal tract organoid model constructed by the construction method, which is used for in-vitro model construction of Bactrian camel intestinal tract related drugs or / and biological products. The construction method can efficiently obtain the Bactrian camel intestinal tract organoid which has self-organization and self-renewal capabilities and highly simulates intestinal epithelial tissue, and realizes stable subculture.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry bioengineering and organoid technology, specifically relating to a method for establishing and applying a Bactrian camel intestinal organoid model. Background Technology

[0002] Bactrian camels (Camelus bactrianus) possess unique physiological adaptations, including tolerance to roughage, hunger, thirst, and extreme temperatures. Their intestinal structure, function, and metabolic characteristics significantly differ from those of ruminants and monogastric animals. The Bactrian camel's intestine is not only the core site for nutrient digestion and absorption but also a crucial organ for maintaining water and electrolyte balance and adapting to extreme environments. It also undertakes complex immune defense functions, playing a vital role in ensuring the health of the camel herd and the safe production of camel milk, camel meat, and other livestock products. However, due to the long growth cycle, high feeding costs, and complex genetic background of Bactrian camels, coupled with the lack of effective in vitro research models, basic and applied research on the regulation of Bactrian camel intestinal development, nutrient metabolism mechanisms, gut microbiota interactions, and the prevention and control of intestinal diseases is severely lagging, hindering the healthy and sustainable development of the Bactrian camel industry.

[0003] Organoid technology, as a rapidly developing in vitro three-dimensional culture system in recent years, can construct miniature organ-like structures with self-renewal and multi-directional differentiation capabilities by simulating the in vivo stem cell microenvironment. It offers unparalleled advantages over traditional two-dimensional cell culture and animal models in preserving the composition, spatial structure, and physiological function of the source tissue cells. Currently, this technology has been widely applied in intestinal research in species such as humans, mice, pigs, cattle, and chickens, successfully used to elucidate intestinal epithelial development, host-microbe interactions, nutrient transport, intestinal infection mechanisms, and drug screening, providing a powerful technical platform for intestinal physiological and pathological research. However, due to the unique characteristics of Bactrian camel intestinal tissue, existing organoid culture protocols based on other species cannot be directly applied; therefore, it is necessary to establish organoid models specifically tailored to the biological characteristics of the Bactrian camel intestine.

[0004] While existing technologies have reported on the culture of intestinal organoids from ruminants (such as cattle and sheep), the culture systems and conditions (such as combinations of growth factors and small molecule inhibitors) are optimized based on the physiological parameters of monogastric or polygastric animals, failing to fully support the normal growth and differentiation of Bactrian camel intestinal organoids. Therefore, developing a method for establishing organoids suitable for Bactrian camel intestinal tissue, effectively maintaining the activity of intestinal stem cells, and reproducing the diversity and functional characteristics of intestinal epithelial cells is of urgent practical need and significant scientific value for filling the technological gap in Bactrian camel intestinal research and promoting basic theoretical research and industrial application technology development in camel science.

[0005] In summary, there is an urgent need in this field for an organoid model establishment method specifically optimized for the intestinal biological characteristics of Bactrian camels, in order to solve the problem of the lack of in vitro research systems for Bactrian camels' specific intestines in the existing technology, and to provide reliable technical support for the regulation of Bactrian camel intestinal nutrition and metabolism, evaluation of intestinal probiotic function, research on the pathogenesis of intestinal diseases, and development of novel feed additives. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for establishing and applying a Bactrian camel intestinal organoid model, which addresses the shortcomings of the prior art. This method can efficiently obtain Bactrian camel intestinal organoids with self-organization and self-renewal capabilities and highly simulate intestinal epithelial tissue, and achieve stable passage culture.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for establishing a Bactrian camel intestinal organoid model, the method being as follows: S1. Intestinal tissue sampling: Obtain intestinal tissue from Bactrian camels; the intestinal tissue from Bactrian camels is placed in ice bath DMEM / F12 medium containing double antibiotics; S2. Tissue pretreatment: Scrape off the intestinal mucosal mucus of the Bactrian camel intestinal tissue obtained in S1, rinse with phosphate buffer, and digest in ethylenediaminetetraacetic acid cell dissociation solution to obtain pretreated intestinal tissue. S3. Crypt Collection: The pretreated intestinal tissue obtained in S2 was rinsed with phosphate buffer, and crypt cells were scraped off to obtain crypt cells. S4. Crypt cell seeding: Add DMEM / F12 medium to the crypt cells obtained in S3 to suspend the cells, sieve, collect the filtrate, centrifuge, discard the supernatant, and resuspend the cells in DMEM / F12 medium containing bovine serum albumin to obtain a cell suspension; add matrix gel to the obtained cell suspension, mix well, seed, and culture to obtain dome-shaped solidified intestinal organoids; S5. Organoid culture: Add growth medium to the dome-shaped solidified intestinal organoids obtained in S4. After culture, a Bactrian camel intestinal organoid model is obtained. S6. Passaging of organoids: Passaging the Bactrian camel intestinal organoid model obtained in S5, discarding the growth medium, blowing the dome with pre-cooled phosphate buffer, digesting, dispersing by blowing, centrifuging, resuspending in DMEM / F12 medium containing bovine serum albumin, and repeating steps S4 and S5 to obtain the Bactrian camel intestinal organoid model.

[0008] Preferably, the Bactrian camel intestinal tissue in S1 is the jejunum or ileum, with a length of 1cm to 2cm.

[0009] Preferably, the number of rinsing cycles in S2 is 6 to 10; the molar concentration of the ethylenediaminetetraacetic acid cell dissociation solution is 5 mmol / L; and the digestion conditions are: digestion for 25 minutes at a temperature of 4°C and a rotation speed of 20 rpm.

[0010] Preferably, the rinsing is performed twice in S3.

[0011] Preferably, the sieve aperture in S4 is 70 μm; the centrifugation conditions are: centrifugation at 200 g for 5 min; the concentration of the cell suspension is 1 × 10⁻⁶. 5 Cells / mL; the volume ratio of the cell suspension to the matrix gel is 1:1; the culture conditions are: cultured at 37℃ for 20 min; Preferably, the growth medium in S5 is prepared by adding the following raw materials to IntestiCult™ organoid growth medium in the following final amounts: Y-27632 10 μM, Adezmapimod 10 μM, Retinoic acid 10 μM, Bactrian camel serum 0.5 v / v%, 100× penicillin-streptomycin-amphoteric acid B mixed solution 1 v / v; the culture conditions are: the medium is changed every 2 days at a temperature of 37°C and 5% CO2; the diameter of the Bactrian camel intestinal organoid model is 100 μm to 150 μm.

[0012] Preferably, the mass fraction of bovine serum albumin in the DMEM / F12 culture medium containing bovine serum albumin in S4 and S6 is 1%; the digestion time in S6 is 3 min to 5 min.

[0013] The present invention also provides the application of the Bactrian camel intestinal organoid model constructed by the above construction method, wherein the Bactrian camel intestinal organoid model is used for the in vitro model construction of Bactrian camel intestinal-related drugs and / or biological products.

[0014] Compared with the prior art, the present invention has the following advantages: This invention addresses the shortcomings of existing livestock intestinal research models and the lack of organoid technology for the unique Bactrian camel species by providing a method for establishing a Bactrian camel intestinal organoid model. This method can efficiently obtain Bactrian camel intestinal organoids with self-organization and self-renewal capabilities that highly simulate intestinal epithelial tissue, and achieve stable passage culture.

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 These are inverted optical microscope images of the experimental and control groups of Bactrian camel intestinal organoids cultured for 24h, 48h, and 72h, respectively, in Example 2 of this invention. Figure 2 Figure 2 shows the cell type identification diagram of Bactrian camel intestinal organoids in Embodiment 2 of the present invention; wherein, Figure A is the t-SNE dimensionality reduction visualization analysis of single-cell transcriptome of Bactrian camel intestinal organoids; Figure B is the comparison of the composition ratio of cell types of Bactrian camel intestinal organoids; and Figure C is the expression analysis of characteristic genes of various cells in Bactrian camel intestinal organoids. Figure 3 These are bright-field images obtained by optical microscope after 48h and 72h of culture of Bactrian camel intestinal organoids in Example 2 of the present invention. Detailed Implementation

[0017] Example 1

[0018] This embodiment describes a method for establishing a Bactrian camel intestinal organoid model. The method is as follows: S1. Intestinal tissue sampling: Select healthy adult Bactrian camels and surgically obtain 2cm length of Bactrian camel jejunal tissue. Immediately place it in ice bath DMEM / F12 medium containing double antibiotics (penicillin 100U / mL, streptomycin 100μg / mL) and transport it back to the laboratory for processing within 2 hours. S2. Tissue pretreatment: The intestinal mucosal mucus of the Bactrian camel intestinal tissue obtained in S1 was gently scraped off with a glass slide. After rinsing 8 times with phosphate-buffered saline (PBS), the tissue was placed in EDTA cell dissociation solution with a molar concentration of 5 mmol / L (EDTA was added to PBS buffer, and the final concentration of EDTA was 5 mmol / L). The tissue was digested for 25 min at a temperature of 4℃ and a rotation speed of 20 rpm to obtain the pretreated intestinal tissue. S3. Crypt Collection: The pretreated intestinal tissue obtained in S2 was rinsed twice with PBS. Crypt cells were then gently scraped from the mucosal side using a sterile scalpel to obtain crypt cells. S4. Crypt cell seeding: Add DMEM / F12 medium to the crypt cells obtained in S3 to suspend the cells. After passing the cells through a 70 μm cell sieve, collect the filtrate. Centrifuge at 200 g for 5 min, discard the supernatant, and resuspend the cells in DMEM / F12 medium containing 1% bovine serum albumin to a concentration of 1 × 10⁻⁶ cells / mL. 5 After obtaining cells per mL, a cell suspension was obtained; Matrigel was added to the obtained cell suspension, mixed well, and 50 μL was seeded into a 24-well plate preheated at 37°C. After culturing at 37°C for 20 min, dome-shaped solidified intestinal organoids were obtained. The volume ratio of the cell suspension to the matrix gel is 1:1; S5. Organoid culture: Add 700 μL of growth medium to each well of the dome-shaped solidified intestinal organoids obtained in S4, and incubate them in an incubator at 37℃ and 5% CO2. Replace the medium with fresh medium on the 3rd day of culture, and then replace the medium every 2 days thereafter to obtain the Bactrian camel intestinal organoid model. The diameter of the Bactrian camel intestinal organoid model is 130 μm; The growth medium was prepared by adding the following ingredients to IntestiCult™ Organoid Growth Medium (Human), STEMCELL Technologies, #06010, in the following final amounts: Y-27632 10 μM, Adezmapimod 10 μM, Retinoic acid 10 μM, Bactrian camel serum 0.5 v / v%, and 100× penicillin-streptomycin-amphotericidal B mixed solution 1 v / v%. S6. Passaging of organoids: The Bactrian camel intestinal organoid model obtained in S5 was passaged. After the growth medium was discarded, the dome was blown with 1 mL of phosphate buffer pre-cooled at 4°C. After digestion with TrypLE Express (Thermo Fisher Scientific (China) Co., Ltd.) for 4 min, it was dispersed into single cells or small cell clusters by blowing. It was centrifuged at 200g for 5 min and resuspended in DMEM / F12 medium containing 1% bovine serum albumin. Steps S4 and S5 were repeated to obtain the Bactrian camel intestinal organoid model.

[0019] The condition parameters for this embodiment are: In step S1, the jejunal tissue of the Bactrian camel can also be ileal tissue, with a length of 1cm to 2cm; The number of rinsing cycles in step S2 can be 6 to 10. In step S5, the diameter of the Bactrian camel intestinal organoid model can be 100μm to 150μm. The digestion time in step S6 can be 3 to 5 minutes. Example 2 This embodiment is a functional integrity test of the Bactrian camel intestinal organoid model constructed in Example 1.

[0020] (1) Establishment of Bactrian camel intestinal organoid model: Fresh Bactrian camel jejunal tissue was collected, and crypt cells were collected according to the method described in Example 1. The cells were resuspended in 50% Matrigel and cultured in 24-well cell culture plates. After Matrigel solidification, the experimental group was given IntestiCult™ organoid growth medium (human) containing 10 μM Y-27632, 10 μM Adezmapimod, 10 μM Retinoic acid, 0.5 v / v% Bactrian camel serum, and 1 v / v% of a 100× penicillin-streptomycin-amphotericidal B mixture. The control group's IntestiCult™ organoid growth medium (human) contained only 10 μM Y-27632 and 1 v / v% of a 100× penicillin-streptomycin-amphotericidal B mixture. After being cultured in an incubator at 37℃ and 5% CO2 for 24h, 48h, and 72h, the intestinal organoids of Bactrian camels were observed under an inverted optical microscope. Normally growing Bactrian camel intestinal organoids were collected, fixed with 4% paraformaldehyde for 24h, and then paraffin sections were prepared and stained with hematoxylin and eosin (HE). The morphology of the intestinal organoid tissues of Bactrian camels was observed under an optical microscope.

[0021] The results are as follows Figure 1 As shown in the figure, the scale bar is 100 μm. After 72 h of culture, normally growing vesicle-like organoids can be observed in the culture plate of the experimental group, while the crypt cells in the culture plate of the control group failed to differentiate and proliferate normally to form organoids. Histological observation revealed that the Bactrian camel intestinal organoids have complete epithelial tissue morphology. The above results show that the present invention has successfully established a Bactrian camel intestinal organoid model, and the model can simulate the three-dimensional structure of the Bactrian camel intestinal mucosal epithelium.

[0022] (2) Identification of cell types of intestinal organoids from Bactrian camels: Intestinal organoids from normally growing Bactrian camels were collected, and nuclei were extracted. Single-cell nuclear transcriptome sequencing was performed using the 10X Genomics platform. After quality control filtering, the sequences were aligned to the Bactrian camel reference genome using Cell Ranger to generate a gene counting matrix. Subsequently, Seurat was used for nuclear quality control, removing low-quality nuclei, twin nuclei, and nuclei with a high proportion of mitochondria. The data were then standardized, normalized, and screened for highly variable genes. After dimensionality reduction using PCA, UMAP was used for visualization, and nuclear clustering was performed based on the Louvain algorithm. Cell type annotation was performed using differentially expressed genes in each cell cluster combined with known cell type marker genes.

[0023] The results are as follows Figure 2As shown in Figure A, the t-SNE dimensionality reduction visualization analysis of single-cell transcriptomes of Bactrian camel intestinal organoids is presented. The horizontal and vertical axes represent the first and second principal components after t-SNE dimensionality reduction, respectively. Each point represents a cell, and a total of 7 cell clusters were identified. Figure B shows the comparison of the composition ratio of cell types in Bactrian camel intestinal organoids. Figure C shows the expression analysis of characteristic genes of various cell types in Bactrian camel intestinal organoids. The main cell types include: epithelial cells (characteristic genes: KRT18, KRT20, EPCAM), absorptive intestinal epithelial cells (characteristic genes: VIL1, APOB, FABP1), TA cells (characteristic genes: PCNA, OLFM4, KRT20), goblet cells (characteristic genes: MUC2, FCGBP, TFF3), enteroendocrine cells (characteristic genes: CHGA, CHGB, NEUROD1, PYY), secretory progenitor cells (characteristic genes: SOX9, DII1, Atoh1), and intestinal cluster cells (characteristic genes: POU2F3, GNAT3, TRPM5, CHAT). The above results demonstrate that the Bactrian camel intestinal organoid model established in this invention can highly simulate the cellular heterogeneity of the intestinal mucosal epithelium of Bactrian camels.

[0024] (3) Passage of Bactrian camel intestinal organoids: When the Bactrian camel intestinal organoids grew to a suitable size and structural integrity in the culture system, the organoids and the mixture of matrix gel in the culture wells were collected. The organoids were mechanically broken into cell clumps by pipetting, centrifuged, the supernatant was discarded, and the organoids were resuspended in TrypLE Express. The organoids were digested at 37°C for 2 min, centrifuged again, the supernatant was discarded, and the organoids were resuspended in pre-cooled PBS. The organoids were collected again by centrifugation and resuspended in 50% matrix gel. The organoids were then re-seeded in 24-well cell culture plates at the initial seeding density and placed in a 37°C, 5% CO2 incubator until the matrix gel was completely cured. Subsequently, the experimental group culture medium, which was the same as that of the primary culture, was added, and the cells were continued to be passaged in a 37°C, 5% CO2 incubator. After 72 h of culture, the growth status of the organoids was observed.

[0025] The results are as follows Figure 3 As shown in the figure, the scale bar is 100 μm. After 72 hours of subculturing, normally growing Bactrian camel intestinal organoids can be observed, indicating that the Bactrian camel intestinal organoids established by this method can be subcultured normally.

[0026] In summary, the Bactrian camel intestinal organoid model constructed in this invention has self-organization and self-renewal capabilities and highly simulates the cellular heterogeneity of Bactrian camel intestinal epithelial tissue. It can be used for the in vitro model construction of Bactrian camel intestinal-related drugs and / or biological products.

[0027] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for establishing a Bactrian camel intestinal organoid model, characterized in that, The method is as follows: S1. Intestinal tissue sampling: Obtain intestinal tissue from Bactrian camels; the intestinal tissue from Bactrian camels is placed in ice bath DMEM / F12 medium containing double antibiotics; S2. Tissue pretreatment: Scrape off the intestinal mucosal mucus of the Bactrian camel intestinal tissue obtained in S1, rinse with phosphate buffer, and digest in ethylenediaminetetraacetic acid cell dissociation solution to obtain pretreated intestinal tissue. S3. Crypt Collection: The pretreated intestinal tissue obtained in S2 was rinsed with phosphate buffer, and crypt cells were scraped off to obtain crypt cells. S4. Crypt cell seeding: Add DMEM / F12 medium to the crypt cells obtained in S3 to suspend the cells, sieve, collect the filtrate, centrifuge, discard the supernatant, and resuspend the cells in DMEM / F12 medium containing bovine serum albumin to obtain a cell suspension; add matrix gel to the obtained cell suspension, mix well, seed, and culture to obtain dome-shaped solidified intestinal organoids; S5. Organoid culture: Add growth medium to the dome-shaped solidified intestinal organoids obtained in S4. After culture, a Bactrian camel intestinal organoid model is obtained. S6. Passaging of organoids: Passaging the Bactrian camel intestinal organoid model obtained in S5, discarding the growth medium, blowing the dome with pre-cooled phosphate buffer, digesting, dispersing by blowing, centrifuging, resuspending in DMEM / F12 medium containing bovine serum albumin, and repeating steps S4 and S5 to obtain the Bactrian camel intestinal organoid model.

2. The method for establishing a Bactrian camel intestinal organoid model according to claim 1, characterized in that, The Bactrian camel intestinal tissue mentioned in S1 is either jejunal or ileal tissue; the length of the Bactrian camel intestinal tissue is 1cm to 2cm.

3. The method for establishing a Bactrian camel intestinal organoid model according to claim 1, characterized in that, The number of times S2 is rinsed is 6 to 10; the molar concentration of the ethylenediaminetetraacetic acid cell dissociation solution is 5 mmol / L; the digestion conditions are: shaking digestion for 25 min at a temperature of 4℃ and a rotation speed of 20 rpm.

4. The method for establishing a Bactrian camel intestinal organoid model according to claim 1, characterized in that, The number of rinses in S3 is 2.

5. The method for establishing a Bactrian camel intestinal organoid model according to claim 1, characterized in that, The sieve in S4 has a pore size of 70 μm; the centrifugation conditions are: centrifugation at 200 g for 5 min; the concentration of the cell suspension is 1 × 10⁻⁶. 5 Cells / mL; the volume ratio of the cell suspension to the matrix gel is 1:1; the culture conditions are: cultured at 37℃ for 20 min.

6. The method for establishing a Bactrian camel intestinal organoid model according to claim 1, characterized in that, The growth medium described in S5 is prepared by adding the following raw materials to IntestiCult™ organoid growth medium in the following final amounts: Y-27632 10 μM, Adezmapimod 10 μM, Retinoic acid 10 μM, Bactrian camel serum 0.5 v / v%, 100× penicillin-streptomycin-amphoteric acid B mixed solution 1 v / v; the culture conditions are: the medium is changed every 2 days at a temperature of 37℃ and 5% CO2; the diameter of the Bactrian camel intestinal organoid model is 100 μm to 150 μm.

7. The method for establishing a Bactrian camel intestinal organoid model according to claim 1, characterized in that, The mass fraction of bovine serum albumin in the DMEM / F12 medium containing bovine serum albumin described in S4 and S6 is 1%; the digestion time in S6 is 3 min to 5 min.

8. An application of a Bactrian camel intestinal organoid model constructed by the construction method according to any one of claims 1-7, characterized in that, The Bactrian camel intestinal organoid model is used for the in vitro model construction of Bactrian camel intestinal-related drugs and / or biological products.

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