Construction and culture method of normal cervix organ

Abstract

The invention relates to the technical field of biomedicine, and discloses a method for constructing and culturing a normal cervical organ, which provides powerful technical support for exploring the pathogenesis of HPV (human papillomavirus) infected normal cervical epithelium.

Description

Technical Field

[0001] The present invention relates to the biomedical field technology, and specifically refers to a method for constructing and culturing normal cervical organoids. Background Art

[0002] Organoids are microscopic three-dimensional structures that can self-assemble when pluripotent stem cells (PSC) and adult stem cells (ASC) are cultured in vitro. Organoids can simulate human organs in structure and function, have genomic stability during long-term in vitro expansion, and have good application potential in regenerative medicine, gene editing, precision medicine, organ development, disease modeling, etc. Organoid models retain the original characteristics of tissues and can more accurately reflect the human body's response to pathogens and drugs. These advantages highlight the great potential of organoids in personalized treatment, especially in predicting drug responses and simulating the interaction between the human body and pathogenic microorganisms.

[0003] Cervical cancer is one of the malignant tumors that seriously threaten women's health. Persistent infection with high-risk HPV is an important cause of the occurrence and development of cervical cancer. Therefore, establishing a scientific and rigorous human normal cervical organoid model can provide strong technical support for exploring HPV infection of normal cervical epithelium and its progression to cancer. At present, there is no mature method for the construction and cultivation of human normal cervical epithelial organoids. Therefore, filling this technical gap will greatly promote the research of cervical-related diseases.

[0004] In view of this, we proposed a method for constructing and culturing normal cervical organoids. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing and culturing normal cervical organoids. The present application also discloses a tissue dissociation solution for dissociating normal cervical tissue, which can fully dissociate the tissue into single cells and improve the cell yield.

[0006] To achieve the above object, the present invention provides the following technical solutions: A culture medium for normal cervical organoids comprises a basic culture medium Advanced DMEM / F12 and specific added factors; the specific added factors comprise: GlutaMaxTM, HEPES, penicillin / streptomycin / amphotericin B, N-acetyl-L-cysteine ​​(NAC), Nicotinamide, A83-01, Y-27632, Human Noggin, Human EGF, Forskolin, Fibroblast growth factor 7, Fibroblast growth factor 10, R-spondin-1, and SB202190.

[0007] Preferably, the concentrations of the components of the specific additive factors are as follows: 1X GlutaMaxTM, 10mM HEPES, 1Xpenicillin / streptomycin / amphotericin B, 10mM Nicotinamide, 1mM N-acetyl-L-cysteine ​​(NAC), 2μM A83-01, 10μM Y-27632, 10uM Forskolin, 100ng / ml Human Noggin, 50ng / ml Human EGF, 200ng / mL Fibroblast growth factor 7, 200ng / mL Fibroblastgrowth factor 10, 10uM R-spondin-1, 1uM SB202190. The concentrations of the components of the specific additive factors are based on their concentrations in the basal culture medium.

[0008] A method for constructing a normal cervical organoid comprises the following steps: 1) Sample processing: After the sample is removed from the body, it is immediately placed in tissue preservation solution and transported to the laboratory on ice. After the tissue is washed with a compound washing solution, the normal cervical tissue block is cut into small pieces; 2) Enzymatic hydrolysis: Transfer the chopped cervical tissue into tissue dissociation solution and digest it to fully dissociate the tissue into a single cell suspension; 3) Red fine sieving: terminate the enzymatic hydrolysis process. Filter the cell suspension after lysis through a cell filter, wash the cell filter thoroughly, collect the filtrate and centrifuge it, remove the supernatant, and obtain high-purity normal cervical epithelial cells; 4) Red blood cell lysis: Add red blood cell lysis buffer to the cell pellet, place on ice for 5 minutes to lyse the red blood cells, and centrifuge again; 5) Plate culture: The cell pellet is pre-suspended into a cell suspension, the cell suspension is resuspended with Matrigel, and plated into an untreated well plate. After the matrix gel solidifies, the complete culture medium as described in claim 1 is added, and the culture is carried out in an incubator.

[0009] Preferably, in step 1), during the sample processing, the composite washing solution comprises: pre-cooled PBS + 100Xpenicillin / streptomycin / amphotericin B (tertiary antibodies), and the specimens treated with the composite washing solution can effectively avoid contamination.

[0010] Preferably, the tissue dissociation solution in step 2) comprises Advanced DMEM / F12 with a final concentration of 1X, 1X penicillin / streptomycin / amphotericin B, collagenase I with a final concentration of 2 mg / mL, neutral proteinase II with a final concentration of 2 mg / mL, DNase I with a final concentration of 0.4 mg / mL, and Y-27632 with a final concentration of 10 μM.

[0011] Preferably, in step 3), the required cell filter specification is 70 um; the centrifugal conditions are: centrifugal speed is 400G, centrifugal temperature is 4°C, and centrifugal time is 5 minutes.

[0012] Preferably, in step (5), the cell pellet is resuspended on ice with Advanced DMEM / F12, 5% FBS solution, and the ratio of solution to matrix gel is 1:4; after the matrix gel is spread on the well plate, the well plate is placed in a 37°C incubator for 30 minutes. After the matrix gel solidifies, the complete culture medium as described in claim 1 is added; the culture medium is replaced every 7 days.

[0013] By means of the above technical solution, the present invention provides a method for constructing and culturing normal cervical organoids, which has at least the following beneficial effects: (1) The culture system of the present invention can improve the success rate of constructing normal human cervical organoids and ensure the activity of organoids. It can be stably passaged, cryopreserved, and revived. It is also suitable for long-term stable culture of normal cervical organoids and expansion of organoid banks. It is an ideal culture system.

[0014] (2) The present invention discloses a tissue dissociation solution for dissociating normal cervical tissue samples. The tissue dissociation solution can better maintain cell viability, allow cells to be fully released during the digestion process, increase the yield of live cells, and significantly reduce costs.

[0015] (3) The composite washing solution of the present invention can effectively reduce the risk of microbial contamination in gastric cancer culture and improve the success rate and survival rate of normal human cervical organoid culture.

[0016] (4) The present invention simulates the in vivo growth environment to the greatest extent possible and maintains the morphological structure and genetic characteristics of the primary tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application: Figure 1 : A low-power microscope example of the organoid obtained in Example 1 of the present invention on the 7th day of the second generation culture; Figure 2 : A low-power microscope example of the organoid obtained in Example 1 of the present invention on the 14th day of the second generation culture; Figure 3 : Illustrations of cystic organoids and solid organoids obtained in Example 1 of the present invention; Figure 4 : HE image of the organoid obtained in Example 1 of the present invention and the tissue of Example 1 Figure 5 : The histochemical map of the organoid obtained in Example 1 of the present invention; DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Mingru Figures 1 to 5 As shown, a method for successfully constructing human normal cervical organoids, the specimens are from surgical specimens. The steps include: 1) Sample processing: After the sample is removed from the body, it is immediately placed in tissue preservation solution and transported to the laboratory on ice. After the tissue is washed with a compound washing solution, the normal cervical tissue block is cut into small pieces; 2) Enzymatic hydrolysis: Transfer the chopped cervical tissue into tissue dissociation solution and digest it to fully dissociate the tissue into a single cell suspension; 3) Red fine sieving: terminate the enzymatic hydrolysis process. Filter the cell suspension after lysis through a cell filter, wash the cell filter thoroughly, collect the filtrate and centrifuge it, remove the supernatant, and obtain high-purity normal cervical epithelial cells; 4) Red blood cell lysis: Add red blood cell lysis buffer to the cell pellet, place on ice for 5 minutes to lyse the red blood cells, and centrifuge again; 5) Plate culture: The cell pellet is pre-suspended into a cell suspension, the cell suspension is resuspended with Matrigel, and plated into an untreated well plate. After the matrix gel solidifies, the complete culture medium as described in claim 1 is added, and the culture is carried out in an incubator.

[0020] The composition of the composite washing solution in step 1) is: pre-cooled PBS + 100X penicillin / streptomycin / amphotericin.

[0021] The tissue dissociation solution in step 2) includes Advanced DMEM / F12 with a final concentration of 1X, 1X penicillin / streptomycin / amphotericin B, 2 mg / mL collagenase I, 2 mg / mL neutral proteinase II, 0.4 mg / mL DNase I, and 10 μM Y-27632 with a final concentration.

[0022] The enzymatic hydrolysis time in step 2) is 2-4 hours; the specific conditions of the digestion treatment are: mechanical dissociation and digestion at 300 rpm / min on a 37°C constant temperature shaker for 1-2 hours, transfer to a six-well plate and manually blow at intervals, and place in a 37°C incubator for digestion until the cervical epithelium is detached in pieces; then add trypsin to fully dissociate the epithelial cells.

[0023] The cell filter required in step 3) is 70um in size; the centrifugal conditions are: centrifugal speed of 400G, centrifugal temperature of 4°C, and centrifugal time of 5 minutes; the washing solution used to wash the filter is 1X Advanced DMEM / F12, 1X penicillin / streptomycin / amphotericin B, and 10% FBS.

[0024] In step 4), the red blood cell lysis solution is 5 mL; the enzymatic termination agent is DMEM culture medium containing 5% FBS; if a large number of red blood cells are observed in the cell precipitate, add 10 mL of red blood cell lysis solution.

[0025] In step 5), the cell pellet is resuspended with Advanced DMEM / F12 and 5% FBS solution, and the ratio of solution to matrix gel is 1:4; after the matrix gel is spread on the well plate, the well plate is placed in a 37°C incubator for 30 minutes. After the matrix gel solidifies, normal cervical organoid complete culture medium is added. The complete culture medium is Advanced DEME / F12 including 1X GlutaMaxTM, 10mM HEPES, 1X penicillin / streptomycin / amphotericin B, 10mM Nicotinamide, 1mM N-acetyl-L-cysteine ​​(NAC), 2μM A83-01, 10μM Y-27632, 10uM Forskolin, 100ng / ml Human Noggin, 50ng / ml Human EGF, 200ng / mL Fibroblast growth factor 7, 200ng / mL Fibroblastgrowth factor 10, 10uM R-spondin-1, and 1uM SB202190.

[0026] According to the above embodiment 1 and Figure 1-Figure 5 It can be seen that according to the construction method and culture mode in Example 1, a culture success rate of more than 90% can be achieved, and the above culture method can maintain the physiological functions and molecular characteristics of the cells, providing reliable research materials. In addition, the organoids still maintain good proliferation activity after multiple passages.

[0027] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0028] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A complete medium, characterized in that: 1X GlutaMaxTM (GlutaMAX™ Supplement), 10mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 1X penicillin / streptomycin / amphotericin B (penicillin-streptomycin-amphotericin B solution), 10mM Nicotinamide, 1mM N-acetyl-L-cysteine ​​(NAC / acetylcysteine), 2μM A83-01, 10μM Y-27632, 10uM Forskolin, 100ng / ml Human Noggin, 50ng / ml Human EGF (human epidermal growth factor), 200ng / mL Fibroblast growth factor 7 (fibroblast growth factor 7), 200ng / mL Fibroblast growth factor 10 (fibroblast growth factor 10), 10uM R-spondin-1, 1uM SB202190 Advanced DEME / F12.

2. A method for constructing a normal human cervical organoid model, characterized in that: The construction method comprises the following steps: (1) Sample processing: After the sample is removed from the body, it is immediately placed in tissue preservation solution and transported to the laboratory on ice. After the tissue is washed, the normal cervical tissue block is cut into small pieces; (2) Enzymatic hydrolysis: The chopped cervical tissue is transferred into a tissue dissociation solution and digested to fully dissociate the tissue into a single cell suspension; (3) Sieving: Terminate the enzymatic hydrolysis process. Filter the cell suspension after lysis through a cell strainer, wash the cell strainer thoroughly, collect the filtrate, centrifuge it, remove the supernatant, and obtain high-purity normal cervical epithelial cells; (4) Red blood cell lysis: Add red blood cell lysis solution to the cell pellet, place on ice for 5 min to lyse the red blood cells, and centrifuge again; (5) Plate culture: The cell pellet is pre-suspended into a cell suspension, the cell suspension is resuspended with Matrigel, and plated into an untreated well plate. After the matrix gel solidifies, the complete culture medium as described in claim 1 is added, and the culture is carried out in an incubator.

3. The method for constructing a normal cervical organoid model according to claim 2, characterized in that: The composite washing solution in step (1) includes: pre-cooled PBS + 100X penicillin / streptomycin / amphotericin B (tertiary antibody).

4. The method for constructing a normal cervical organoid model according to claim 2, characterized in that: The enzymatic solution in step (2) includes: 1X Advanced DMEM / F12, 1X penicillin / streptomycin / amphotericin B, collagenase I with a final concentration of 2 mg / mL, neutral proteinase II with a final concentration of 2 mg / mL, DNase I with a final concentration of 10 μM.

5. The method for constructing a normal cervical organoid model according to claim 2, characterized in that: The enzymatic hydrolysis time in step (2) is 2-4 hours; the specific conditions of the digestion treatment are: mechanical dissociation and digestion at 300 rpm / min on a 37°C constant temperature shaker for 1-2 hours, transfer to a six-well plate and manually blow at intervals, and place in a 37°C incubator for digestion until the cervical epithelium is detached in pieces; then add pancreatic enzyme to fully dissociate the epithelial cells.

6. The method for constructing a normal cervical organoid model according to claim 2, characterized in that: The cell filter required in step (3) is 70 um in size; the centrifugal conditions are: centrifugal speed of 400 G, centrifugal temperature of 4° C., and centrifugal time of 5 minutes.

7. The method for constructing a normal cervical organoid model according to claim 2, characterized in that: In the step (5), the cell precipitate is resuspended with Advanced DMEM / F12 and 5% FBS solution, and the ratio of the solution to the matrix gel is 1:4; after the matrix gel is spread on the well plate, the well plate is placed in a 37°C incubator for 30 minutes. After the matrix gel solidifies, the complete culture medium as described in claim 1 is added.

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