A human embryonic lung alveolus organoid culture medium formulation

Abstract

The present application relates to the technical field of organoid culture, in particular to a kind of human embryo alveolus organoid culture medium formula, including basic culture medium, basic additive, core growth factor, classic small molecule and novel small molecule additive combination;The basic culture medium is Advanced DMEM / F-12, and the working concentration is 1X;The basic additive includes HEPES, GlutaMAX, N-acetyl cysteine, B-27 additive (without vitamin A) and N-2 additive;The core growth factor includes human Noggin recombinant protein, human FGF-10 recombinant protein, human FGF-7 recombinant protein and human EGF recombinant protein;The classic small molecule includes CHIR99021 and Y-27632;The novel small molecule additive combination is GSK126 and AICAR.The beneficial effects of the present application are: long-term stable amplification is realized: through the synergistic effect of novel small molecule additive combination (GSK126 and AICAR), in combination with the optimized collocation of basic additive, core growth factor and classic small molecule, the amplification generation of human embryo alveolus organoid is significantly prolonged.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a human embryonic alveolar organoid culture medium formulation. Background Technology

[0002] Organoids are three-dimensional cellular aggregates formed in vitro, mimicking the development of organs in vivo, based on stem cells or tissue progenitor cells. They possess similar structure and function to their corresponding in vivo organs. Human embryonic alveolar organoids, as important models for studying lung development, disease mechanisms, and drug screening, have broad application prospects in the biomedical field.

[0003] However, the long-term culture of human embryonic alveolar organoids currently faces numerous challenges. The most critical issue is the gradual decline in the proportion of alveolar type 2 cells (AT2 cells) during long-term culture, making it difficult to maintain the stemness of alveolar progenitor cells. This leads to weakened organoid proliferation and reduced functional stability, severely limiting their application in large-scale biobank construction and aging research. Existing culture medium formulations primarily focus on short-term promotion of organoid formation, failing to effectively address the issues of maintaining stemness and stabilizing the proportion of specific cell types during long-term culture.

[0004] Therefore, developing a culture medium formulation that can achieve long-term stable expansion of human embryonic alveolar organoids and maintain a high proportion of AT2 cells is of great practical significance. Summary of the Invention

[0005] To address the problems mentioned in the background art, the present invention aims to provide a human embryonic alveolar organoid culture medium formulation.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] A human embryonic alveolar organoid culture medium formulation, characterized by comprising a basal culture medium, basic additives, core growth factors, classic small molecules, and a combination of novel small molecule additives; the basal culture medium is Advanced DMEM / F-12 at a working concentration of 1X; the basic additives include HEPES, GlutaMAX, N-acetylcysteine, B-27 additive (without vitamin A), and N-2 additive; the core growth factors include recombinant human Noggin protein, recombinant human FGF-10 protein, recombinant human FGF-7 protein, and recombinant human EGF protein; the classic small molecules include CHIR99021 and Y-27632; and the combination of novel small molecule additives consists of GSK126 and AICAR.

[0008] Preferably, the working concentrations of each basic additive are as follows: HEPES 10mM, GlutaMAX 2mM, N-acetylcysteine ​​1.25mM, B-27 additive (without vitamin A) 1X, and N-2 additive 1X.

[0009] Preferably, the working concentrations of each core growth factor are as follows: 100 ng / mL for recombinant human Noggin, 100 ng / mL for recombinant human FGF-10, 50 ng / mL for recombinant human FGF-7, and 50 ng / mL for recombinant human EGF.

[0010] Preferably, the working concentrations of each classic small molecule are as follows: CHIR99021 is 3 μM, Y-27632 is 10 μM, and Y-27632 is used only within the first 2-3 days after passage or dissociation.

[0011] The human embryonic alveolar organoid culture medium formulation is characterized in that the working concentrations of each component in the novel small molecule additive combination are as follows: GSK126 is 1 μM and AICAR is 100 μM.

[0012] The aforementioned human embryonic alveolar organoid culture medium formulation is used for the long-term stable expansion of human embryonic alveolar organoids, maintenance of alveolar progenitor cell stemness, or maintenance of the SFTPC+AT2 cell ratio.

[0013] The culture of the human embryonic alveolar organoids includes the following steps:

[0014] (1) Sample processing: After sterilization, human embryonic alveolar tissue samples were washed with pre-cooled D-Hanks, cut into small pieces, and centrifuged to remove the supernatant;

[0015] (2) Tissue digestion: Add tissue digestion solution to the tissue precipitate and digest at 37°C for 5-20 minutes. Stop digestion when most cells are observed to have detached from the tissue block under a microscope.

[0016] (3) Cell purification: After digestion is terminated, the cells are centrifuged, the cell pellet is collected, and purified cells are obtained after washing, filtration, and red blood cell lysis (if necessary);

[0017] (4) Inoculation and culture: Mix the purified cells thoroughly with Matrigel and inoculate them into a preheated culture plate. After Matrigel solidifies, add the culture medium described in any one of claims 1-5 and culture in a cell culture incubator at 37°C. Change the medium every 2-3 days.

[0018] The beneficial effects of this invention are:

[0019] Achieving long-term stable expansion: Through the synergistic effect of a novel combination of small molecule additives (GSK126 and AICAR), combined with the optimized combination of basic additives, core growth factors and classic small molecules, the number of expansion generations of human embryonic alveolar organoids was significantly extended, solving the problem of weakened organoid proliferation capacity during long-term culture.

[0020] Maintaining a high proportion of AT2 cells: It effectively maintained a high proportion of SFTPC+AT2 cells during long-term culture, overcoming the technical bottleneck of declining AT2 cell proportion in existing technologies, and ensuring the functional stability of organoids.

[0021] Locking in the state of alveolar progenitor cells: By "locking" alveolar progenitor cells in a state of high proliferation and low spontaneous differentiation, a stable cell source is provided for the study of lung development mechanisms and the construction of disease models.

[0022] It has a wide range of applications: it is suitable for multiple fields such as building large-scale biobanks, conducting aging research, and drug screening, and has important scientific research value and application prospects.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0025] The present invention is illustrated below with specific embodiments, which are not intended to limit the scope of the invention.

[0026] A human embryonic alveolar organoid culture medium formulation, characterized in that:

[0027] The composition includes a basal culture medium, basic additives, core growth factors, and a combination of classic and novel small molecule additives. The specific settings of each component are as follows:

[0028] Basic culture medium: AdvancedDMEM / F-12 was used as the basic culture medium at a working concentration of 1X to provide a basic nutritional environment for organoid growth.

[0029] Basic additives include HEPES, GlutaMAX, N-acetylcysteine, B-27 additive (without vitamin A), and N-2 additive. HEPES is administered at a working concentration of 10 mM to maintain pH stability in the culture medium; GlutaMAX is administered at a working concentration of 2 mM as a stable glutamine substitute to support cell metabolism; N-acetylcysteine ​​is administered at a working concentration of 1.25 mM to exert antioxidant effects and reduce oxidative stress damage to cells; B-27 additive (without vitamin A) is administered at a working concentration of 1X to provide key hormones, vitamins, and trace elements; and N-2 additive is administered at a working concentration of 1X to provide essential components such as transferrin, insulin, and selenium.

[0030] Core growth factors include recombinant human Noggin, recombinant human FGF-10, recombinant human FGF-7, and recombinant human EGF. The working concentration of recombinant human Noggin (100 ng / mL) inhibits BMP signaling and promotes foregut / lung fate; the working concentration of recombinant human FGF-10 (100 ng / mL) acts as a key alveolar progenitor mitogen, promoting alveolar progenitor cell proliferation; the working concentration of recombinant human FGF-7 (50 ng / mL) promotes alveolar epithelial proliferation and branching morphogenesis; and the working concentration of recombinant human EGF (50 ng / mL) promotes epithelial cell proliferation.

[0031] Classic small molecules include CHIR99021 and Y-27632. CHIR99021, at a working concentration of 3 μM, can activate Wnt signaling, which is crucial for the development and maintenance of alveolar 2 cells (AT2). Y-27632, at a working concentration of 10 μM, should be used strictly within the first 2-3 days after passage or dissociation to inhibit apoptosis and improve cell survival efficiency.

[0032] A novel combination of small molecule additives: GSK126 and AICAR, with a working concentration of 1 μM for GSK126 and 100 μM for AICAR. GSK126 is a highly selective and potent EZH2 inhibitor that influences cell fate through epigenetic regulation; AICAR is an AMPK activator that regulates cellular energy metabolism and growth. The combined use of these two additives constitutes the core patent point of this invention, synergistically addressing technical bottlenecks in long-term culture.

[0033] Synergistic mechanism

[0034] The core innovation of this invention lies in the combined use of a novel combination of small molecule additives (GSK126 and AICAR), whose synergistic mechanism is as follows:

[0035] GSK126 promotes cell self-renewal and delays cell senescence and spontaneous differentiation by inhibiting histone H3K27 trimethyltransferase EZH2 and opening gene loci related to the maintenance and proliferation of alveolar progenitor cells stemness.

[0036] AICAR activates AMPK by mimicking AMP, guiding the metabolic state of cells toward a direction more conducive to proliferation and survival, enhancing energy homeostasis, and synergistically promoting proliferation with FGF signaling.

[0037] The two small molecules mentioned above, together with basic additives, core growth factors, and classic small molecules, work together to significantly extend the number of generations of alveolar organoids and maintain a higher proportion of alveolar 2 cells. This successfully solves the technical bottleneck of the decline in the proportion of SFTPC+AT2 cells during long-term culture, and "locks" alveolar progenitor cells in a highly proliferating state that is not prone to spontaneous differentiation.

[0038] Specific implementation steps:

[0039] 1. During preparation, strictly adhere to aseptic techniques. After thorough mixing of all components, store at 4°C for short-term storage or -20°C for long-term storage. Before use, allow to return to room temperature and mix again. Y-27632 should be added to the culture medium within the first 2-3 days after subculturing or dissociation.

[0040] 2. Procedures for culturing human embryonic alveolar organoids

[0041] (1) Preparation: Clean the laminar flow hood and prepare the microscope, centrifuge, cell culture incubator and other experimental instruments in advance; place sterile pipette tips, pipettes, sterile centrifuge tubes, surgical instruments (sterilized by autoclaving one day in advance), 70-100μm filter sieves, culture dishes (preheated in a 37℃ incubator in advance) and other consumables in the laminar flow hood and turn on the UV sterilizer for at least 30 minutes; prepare the tissue preservation solution, the organoid culture medium of this invention, D-Hanks (add 1%-2% penicillin and streptomycin to the final concentration, pre-cooled in a 4℃ refrigerator in advance), anti-adhesion agent, tissue digestion solution, red blood cell lysis solution, Matrigel (taken out of the -20℃ refrigerator two hours in advance and thawed in a 4℃ refrigerator) and other reagents, and ensure that the reagents are complete and sufficient. The formula for the tissue preservation solution is: AdvancedDMEM / F12+Y27632 (10μM)+1% penicillin and streptomycin.

[0042] (2) Sample collection: After obtaining human embryonic alveolar tissue samples clinically, transfer them as quickly as possible to sterile EP tubes containing sufficient tissue preservation solution, strictly following aseptic procedures, and transport them at low temperature or store them briefly at 4°C. It should be noted that research involving human tissue samples must comply with all relevant institutional and government regulations, and informed consent must be obtained from all subjects before collecting human tissue samples.

[0043] (3) Sample processing: Before extracting primary cells, carefully observe the size, texture and color of the sample, take pictures and record them in detail for later analysis; after disinfecting the EP tube containing the tissue sample with alcohol, transfer it to the laminar flow hood, take the sample out into a 10cm culture dish, take an appropriate amount of tissue for primary cell extraction, wash with pre-cooled D-Hanks to remove blood clots, wash repeatedly for about 3-5 times until the washing solution is clear; collect an appropriate amount of tissue into a 1.5mL EP tube, cut the tissue into small pieces with surgical scissors and transfer it into a 15mL centrifuge tube, add 5-10mL D-Hanks and rinse again, centrifuge at 300xg for 3min, and remove the supernatant.

[0044] (4) Tissue digestion: Add an appropriate amount of tissue digestion solution to the centrifuged tissue precipitate and mix well (the volume of the digestion solution should be 3-5 times the volume of the tissue). Digest at 37°C for 5-20 minutes, taking it out every 3-5 minutes to observe the degree of digestion. When most cells are observed to have detached from the tissue block under a microscope, digestion can be stopped. After tissue digestion, the centrifuge tubes and pipette tips should be rinsed with an anti-adhesion agent during all operations to avoid cell loss.

[0045] (5) Termination of digestion and cell purification: Add 2 volumes of D-Hanks and mix well to terminate digestion. Centrifuge at 300xg for 5 min and discard the supernatant. Add 5-10 mL of D-Hanks again and mix well. Filter through a 70 μm filter and centrifuge at 300xg for 3 min and discard the supernatant. Add 5 mL of D-Hanks to the cell pellet, pipette and wash, centrifuge at 300xg for 3 min and discard the supernatant. Repeat 1-2 times. If there are too many red blood cells, red blood cell lysis buffer can be used. The lysis time depends on the instructions of different manufacturers and batches of reagents. If there are many red blood cells, the lysis time can be repeated or extended. Strict timing and timely termination are required.

[0046] (6) Inoculation and culture: Take out the Matrigel that has been thawed in the refrigerator at 4°C and place it on ice. Take an appropriate amount of Matrigel and mix it thoroughly with the cell pellet (the inoculation density can be adjusted according to the experimental requirements) to make the Matrigel content ≥70%. The entire process is carried out on ice to avoid the formation of air bubbles in the gel and uneven concentration affecting organoid growth. Inoculate the cells mixed with Matrigel into a cell culture plate preheated at 37°C. Inoculate the gel droplet in the center of the culture well. If it is a 24-well plate, 30-40 μL can be inoculated in each well. After the inoculated culture plate is placed in a cell culture incubator at 37°C and left to stand for 3 min, invert the culture plate for 15-20 min. After Matrigel has completely solidified, take it out and add 500 μL of the organoid culture medium of the present invention to each well. Put it back into the incubator for culture. After that, change the medium every 2-3 days depending on the cell volume and growth status. Track and record the growth status by taking pictures. When the organoids grow to a certain size (generally about 100 μm) or when it is found that the density is too high and not conducive to growth, perform passage treatment.

[0047] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0048] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A culture medium formulation for human embryonic alveolar organoids, characterized in that, The formulation includes a basal culture medium, basic additives, core growth factors, classic small molecules, and a combination of novel small molecule additives. The basal culture medium is Advanced DMEM / F-12 at a working concentration of 1X. The basic additives include HEPES, GlutaMAX, N-acetylcysteine, B-27 additive (without vitamin A), and N-2 additive. The core growth factors include recombinant human Noggin protein, recombinant human FGF-10 protein, recombinant human FGF-7 protein, and recombinant human EGF protein. The classic small molecules include CHIR99021 and Y-27632. The combination of novel small molecule additives consists of GSK126 and AICAR.

2. The human embryonic alveolar organoid culture medium formulation according to claim 1, characterized in that, The working concentrations of each basic additive are as follows: HEPES is 10mM, GlutaMAX is 2mM, N-acetylcysteine ​​is 1.25mM, B-27 additive (without vitamin A) is 1X, and N-2 additive is 1X.

3. The human embryonic alveolar organoid culture medium formulation according to claim 1, characterized in that, The working concentrations of each core growth factor are as follows: recombinant human Noggin 100 ng / mL, recombinant human FGF-10 100 ng / mL, recombinant human FGF-7 50 ng / mL, and recombinant human EGF 50 ng / mL.

4. The human embryonic alveolar organoid culture medium formulation according to claim 1, characterized in that, The working concentrations for each classic small molecule are as follows: CHIR99021 is 3 μM, Y-27632 is 10 μM, and Y-27632 is only used within the first 2-3 days after passage or dissociation.

5. The human embryonic alveolar organoid culture medium formulation according to claim 1, characterized in that, The working concentrations of each component in the novel small molecule additive combination are as follows: GSK126 is 1 μM, and AICAR is 100 μM.

6. The application of the human embryonic alveolar organoid culture medium formulation according to any one of claims 1-5 in the long-term stable expansion of human embryonic alveolar organoids, maintenance of alveolar progenitor cell stemness, or maintenance of the SFTPC+AT2 cell ratio.

7. The application according to claim 6, characterized in that, The culture of the human embryonic alveolar organoids includes the following steps: (1) Sample processing: After sterilization, human embryonic alveolar tissue samples were washed with pre-cooled D-Hanks, cut into small pieces, and centrifuged to remove the supernatant; (2) Tissue digestion: Add tissue digestion solution to the tissue precipitate and digest at 37°C for 5-20 minutes. Stop digestion when most cells are observed to have detached from the tissue block under a microscope. (3) Cell purification: After digestion is terminated, the cells are centrifuged, the cell pellet is collected, and purified cells are obtained after washing, filtration, and red blood cell lysis (if necessary); (4) Inoculation and culture: Mix the purified cells thoroughly with Matrigel and inoculate them into a preheated culture plate. After Matrigel solidifies, add the culture medium described in any one of claims 1-5 and culture in a cell culture incubator at 37°C. Change the medium every 2-3 days.