CN112430568B - Method for feeding epithelial source organoid by umbilical cord mesenchymal stem cells - Google Patents

Method for feeding epithelial source organoid by umbilical cord mesenchymal stem cells Download PDF

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CN112430568B
CN112430568B CN202011333320.0A CN202011333320A CN112430568B CN 112430568 B CN112430568 B CN 112430568B CN 202011333320 A CN202011333320 A CN 202011333320A CN 112430568 B CN112430568 B CN 112430568B
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mesenchymal stem
umbilical cord
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CN112430568A (en
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邱培
朱宇
廖传荣
黄敏
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Accurate International Biotechnology Guangzhou Co ltd
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    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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Abstract

The invention provides a method for feeding epithelial source organoids by umbilical cord mesenchymal stem cells. In the method, an epithelial-derived tissue is located at the center of the structural pattern, and the epithelial-derived tissue is coated with a first gel layer, a second gel layer, and a third gel layer in this order from the inside to the outside, the third gel layer is coated with a liquid layer, and below the third gel layer in the liquid layer is an umbilical cord mesenchymal stem cell feeder layer. According to the invention, the human umbilical cord mesenchymal stem cells are used as a feeder layer and the organoid is cultured in the structural mode, so that on one hand, the culture system contains the minimum components required by culturing the organoid with epithelial sources (including tumors), the application range is wide, on the other hand, tissues with multiple sample sources such as normal epithelia and solid tumors can be cultured, and the culture success rate is high.

Description

Method for feeding epithelial source organoid by umbilical cord mesenchymal stem cells
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a method for feeding epithelial source organoids by umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of pluripotent stem cells derived from early-developing mesoderm and ectoderm, and have the ability to self-renew and differentiate in multiple directions. MSCs currently under investigation are mainly derived from adult bone marrow and umbilical cord blood. For adult bone marrow-derived mesenchymal stem cells, the number and proliferation differentiation potential of the mesenchymal stem cells are reduced with the increase of age, the virus infection rate is high, and the acquisition of the mesenchymal stem cells is limited to a certain extent because bone marrow collection of a donor needs bone marrow puncture. In recent years, a great deal of research shows that a great deal of mesenchymal stem cells also exist in human umbilical cords, and the human umbilical cords have the advantages of wide sources, convenient material taking, relative purity, rich content, low immunogenicity and the like, and gradually replace the mesenchymal stem cells from other sources to become one of the hotspots in the research field of MSCs.
The umbilical cord is a cord-like structure connected between the umbilical part of embryo and placenta, covered with amnion, and contains jelly-like connective tissue derived from mesoderm. A large number of researches prove that the umbilical cord contains abundant mesenchymal stem cells, and the sources of the mesenchymal stem cells are mainly divided into four types: derived from Wharton's jelly; derived from the periphery of umbilical vessels; is derived from cord blood; derived from the intravascular and subcutaneous umbilical vein. Because human umbilical cord mesenchymal stem cells (hucmmesenchymal stem cells, hUCMSCs) have unique advantages and characteristics, the human umbilical cord mesenchymal stem cells are expected to become a good cell source for treating various diseases by cell transplantation, and the advantages are mainly reflected in the following aspects: firstly, the hUCMSCs have strong adhesiveness, can be attached to the wall quickly when low-density cell culture is carried out, have high proliferation speed, can be induced into osteoblasts, adipogenic cells, nerve cells and the like in vitro, and have multidirectional differentiation potential. Secondly, hUCMSCs do not express Major Histocompatibility Complex (MHC) class II antigens and do not express or underexpress multiple co-stimulatory factors associated with transplant immune rejection, such as CD80, CD40, CD46, CD40L, are a class of immunodeficiency cells that have superior proliferative capacity and lower immunogenicity than bone marrow MSCs. The study showed that the percentages of lymphocytes and monocytes in cord blood after separation were 60.56% and 17.93%, respectively, which are significantly more than 46.75% and 10.70% of the bone marrow blood. After separation, the CD34 and CD38 of the cord blood stem cells are obviously more than the bone marrow blood stem cells and are respectively 5.7:1.6 and 2.4: 1.6. Moreover, the umbilical cord after delivery belongs to waste, the material taking is very convenient, and the umbilical cord is not limited by ethical morals; finally, the content of hUCMSCs is very rich, and research shows that 107 primary cells can be obtained by isolated culture in an umbilical cord with the length of 30 cm, the differentiation capacity of the cells is strong, the biological performance is stable, the strong proliferation and differentiation capacities can be still maintained after multiple passages, and sufficient cell sources are provided for scientific research work and clinical treatment. In conclusion, the umbilical cord mesenchymal stem cells have strong advantages in all aspects, so the umbilical cord mesenchymal stem cells have very wide application prospects in the field of cell therapy.
Organoids (organoids) are three-dimensional cell complexes that are structurally and functionally similar to the target organ or tissue, induced by in vitro 3D culture techniques to differentiate stem cells or organ progenitors, have stable phenotypic and genetic characteristics, can be cultured in vitro for long periods of time, and reproduce two events that occur in vivo during formation, i.e., clustering and spatially specific cell lineage commitment of cells of the same type in an adherent manner. Compared with the traditional 2D cell culture mode, the organoid cultured in 3D comprises a plurality of cell types, breaks through the pure physical contact relation among cells, forms more compact biological communication among the cells, realizes the mutual influence, induction and feedback among the cells, develops in a cooperative way and forms a functional mini organ or tissue, can be better used for simulating the generation process and the physiological and pathological states of organ tissues, and has wide application prospect in the aspects of basic research and clinical diagnosis and treatment.
However, the conventional method of culturing mammalian stem cells to form organoids by 3D culture techniques relying on sequential addition of growth factors still has its limitations, such as inaccurate control of organoids and their local environment, and furthermore, the method does not replicate the complex and dynamic microenvironment of organs during their development well, thereby making it difficult to obtain more complete organoids that resemble organ development in vivo. Meanwhile, the difference between the in vitro culture environment and the in vivo of a patient is large, and the intact cell factors suitable for growth and proliferation of the organoid are lacked, so that the organoid is aged and died quickly in the culture process, and the success rate of organoid culture is low finally. Therefore, there is a need to continue to search for new organoid preparation techniques.
Disclosure of Invention
Aiming at the problems of the existing organoid preparation technology, the invention aims to provide a method for feeding epithelial source organoids by umbilical cord mesenchymal stem cells. The technical scheme of the invention is as follows:
in a first aspect, the present invention provides a structural pattern of umbilical cord mesenchymal stem cell feeder epithelial-derived organoids in which an epithelial-derived tissue is located at the center of the structural pattern, and the epithelial-derived tissue is coated with, in order from the inside to the outside, a first gel layer, a second gel layer, and a third gel layer, the third gel layer being coated with a liquid layer, and an umbilical cord mesenchymal stem cell feeder layer located below the third gel layer in the liquid layer.
In a second aspect, the present invention provides a method for feeding umbilical cord mesenchymal stem cells to an epithelial-derived organoid, which utilizes the above structural pattern for culture.
Further, the method comprises the steps of:
step 1, preparing an umbilical cord mesenchymal stem cell feeder layer;
step 2, processing the epithelial source tissue into a cell mass with the cell number of 3-50 cells;
step 3, taking three parts of gel culture medium, and suspending the cell mass obtained in the step 2 by the first part of gel culture medium to obtain cell culture solution; solidifying a second portion of the gel culture medium into a gel drop, then adding the cell culture fluid to the gel drop and further forming a double-layer gel drop comprising a first gel layer and a second gel layer; adding a third gel culture medium to the double-layer gel drops to form a third gel layer;
and 4, adding an umbilical cord mesenchymal stem cell feeder layer into the liquid culture medium, completely soaking the three layers of glue drops obtained in the step 3 into the liquid culture medium, enabling the three layers of glue drops to be located above the feeder layer, and culturing until organoids are formed.
Further, the process for preparing the umbilical cord mesenchymal stem cell feeder layer in the step 1 comprises the following steps: inoculating human umbilical cord mesenchymal stem cells, culturing until the cell confluency is 40-60%, adding 4-10 μ g/ml mitomycin C, treating for 4-8h, and removing mitomycin C.
Preferably, the second portion of gel medium in step 2 is solidified into droplets in a manner that: adding a second portion of gel medium to the center of the inserted cell culture plate, and placing the inserted cell culture plate in a sterile, closed, 37 deg.C, 5% CO2Culturing under inverted condition for 15-30min, and coagulating.
Further, the gel medium comprises the following components in final concentrations: GIBCO serum replacement: 5-30 wt%; EW-7197-HCl: 100-; SB 203580-HCl: 1-40 mu M; y-27632 dihydrochloride: 2-50 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; matrigel: 3-6 mg/ml; the solvent is DMEM/F-12 medium.
Further, the control conditions of culturing to organoid formation in the step 4 are as follows: at 37 ℃ with 5% CO2Culturing under the condition for 4-7 days, and replacing the liquid culture medium every 2-3 days.
Further, the liquid medium comprises the following components at the final concentrations: GIBCO serum replacement: 5-30 wt%; EW-7197-HCl: 100-; SB 203580-HCl: 1-40 mu M; y-27632 dihydrochloride: 2-50 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; the solvent is DMEM/F-12 medium.
In a third aspect, the present invention provides an organoid of epithelial origin obtainable by the above method.
The invention has the beneficial effects that: the culture method of the invention adopts the human umbilical cord mesenchymal stem cells as the feeder layer and the suspension type culture structure mode, on one hand, the culture system contains the minimum components required by the culture of epithelial-derived (tumor-containing) organoids, the application range is wide, on the other hand, tissues from multiple sample sources such as normal epithelia and solid tumors can be cultured, and the culture success rate is higher. The features of the invention are further summarized as follows:
compared with the mesenchymal stem cells of the marrow, the human umbilical cord mesenchymal stem cells have the advantages of wide sources, convenient material taking, relative purity, rich content, low immunogenicity and the like;
the human umbilical cord mesenchymal stem cells adopted by the invention are taken as feeder cells, can secrete various specific cytokines, can better simulate a complex in-vivo environment, and avoids the complexity of manual growth factor adding operation and the uncertainty of contents, thereby being beneficial to the growth and proliferation of organoids, the maintenance of related functions and gene phenotypes;
the suspension type culture structure mode adopted by the invention can avoid the direct interaction of organoid and feeder layer cells to influence the physiological functions of organoid and feeder layer cells, and the cells are arranged in the two layers of gel, thus being more beneficial to the self-organization differentiation of small cell clusters;
the suspension culture method avoids the adherence of the organoid caused by the gravity action in the traditional glue dripping method, and is more beneficial to the later-stage tests such as passage, observation, detection and the like.
Drawings
FIG. 1 is a schematic structural diagram of the structural mode of an organ organoid of umbilical cord mesenchymal stem cell feeder epithelium source in example 1 of the present invention.
FIG. 2 is a top view of the inserting cell culture plate Hanging cell inserts in FIG. 1, and in FIGS. 1-2, 1-liquid culture medium, 2-first gel layer, 3-second gel layer, 4-third gel layer, 5-human umbilical cord mesenchymal stem cell feeder layer, and 6-inserting cell culture plate.
FIG. 3 is a schematic diagram of the tissue morphology structure of lung cancer organoids obtained in example 5 of the present invention.
FIG. 4 is a schematic diagram showing a morphological structure of a tissue of an intestinal cancer organoid obtained in example 6 of the present invention.
FIG. 5 is a schematic diagram of the tissue morphology structure of the breast cancer organoids obtained in example 7 of the present invention.
FIG. 6 is a schematic diagram showing the tissue morphology structure of lung cancer organoids obtained in comparative example 1 of the present invention.
FIG. 7 is a schematic diagram showing a morphological structure of a tissue of an intestinal cancer organoid obtained in comparative example 2 of the present invention.
FIG. 8 is a schematic diagram showing the tissue morphology structure of a breast cancer organoid obtained in comparative example 3 of the present invention.
FIG. 9 is a schematic diagram showing the tissue morphology structure of the lung cancer organoid obtained in comparative example 4 of the present invention.
FIG. 10 is a schematic view showing a morphological structure of a tissue of an intestinal cancer organoid obtained in comparative example 5 of the present invention.
FIG. 11 is a schematic diagram showing the tissue morphology structure of a breast cancer organoid obtained in comparative example 6 of the present invention.
FIG. 12 is a schematic diagram showing the tissue morphology structure of the lung cancer organoid obtained in comparative example 7 of the present invention.
FIG. 13 is a schematic diagram showing the tissue morphology structure of a lung cancer organoid obtained in comparative example 8 of the present invention.
Detailed Description
The inserted cell culture plates used in the practice of the invention were Hanging cell inserts, purchased from Millipore corporation.
The GIBCO serum substitute adopted in the implementation of the invention is KnockOutTMSR-Multi-specifices, available from Thermo Fisher Scientific, Inc.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
As shown in fig. 1 and 2, the present embodiment provides a structural mode of feeding umbilical cord mesenchymal stem cells to an epithelial-derived organoid, in which an insertion cell culture plate handling cellservers is used, the epithelial-derived tissue is located at the center of the structural mode, and the epithelial-derived tissue is sequentially coated with a first gel layer, a second gel layer and a third gel layer in this order from the inside to the outside, the third gel layer is coated with a liquid layer, and the umbilical cord mesenchymal stem cells feeding layer is located below the third gel layer in the liquid layer.
Example 2
The embodiment provides a method for feeding lung cancer organoids by umbilical cord mesenchymal stem cells, which adopts the structural mode of the embodiment 1, and the method comprises the following steps:
step 1, preparing a feeding layer: inoculating human umbilical cord mesenchymal stem cells to a culture dish, culturing until the confluence degree of the cells is 50%, adding 7 mu g/ml of mitomycin C into a human umbilical cord mesenchymal stem cell culture medium, treating for 6h, and removing the mitomycin C to obtain a feeding layer;
step 2, tissue pretreatment: pretreating a fresh-source surgical excision specimen or biopsy tissue, wherein the pretreatment step comprises washing, chopping, digesting tissue blocks, filtering to remove impurities and the like to obtain a cell mass with the cell number of 3-50 cells, and centrifuging to remove a supernatant for later use;
step 3. mu.l of gel medium (containing Matrigel component) was added to the center of the inserted cell culture plate Hanging cell inserts membrane placed upside down in a sterilized plastic box matching the size of the cell culture dish, the box was closed, 5% CO was added at 37 ℃ C2Cultured in an incubator15min, standing for gelation; the gel medium included the following components at the final concentrations: GIBCO serum replacement: 10 wt%; EW-7197-HCl: 1000 nM; SB 203580-HCl: 20 mu M; y-27632 dihydrochloride: 25 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; matrigel: 5 mg/ml; the solvent is DMEM/F-12 culture medium;
step 4. resuspending 5. mu.l of the cell pellet obtained in step 2 in the gel medium (containing Matrigel component), mixing the cell pellet with a pipette, adding the cell pellet to the gel drop solidified by the Hanging cell inserts obtained in step 3 to form a gel drop, closing the box, and adding 5% CO at 37 deg.C2Culturing for 15min in an incubator until the gel is solid for use;
step 5. Add 10. mu.l of the above gel medium (containing Matrigel component) to the solidified double-layered gel drop obtained in step 4 to cover the previous gel drop, close the box, and 5% CO at 37 deg.C2Culturing for 15min in an incubator until the gel is solid for use;
and 6, Hanging the Hanging cell inserts obtained in the step 5 on a culture dish inoculated with the human umbilical cord mesenchymal stem cell feeder layer prepared in the step 1 in a positive mode, and then adding a liquid culture medium into the culture dish to enable the glue drops on the Hanging cell inserts to be fully immersed in the liquid level of the culture medium. Then placing in a constant temperature incubator at 37 ℃ and 5% CO2Culturing under the concentration; the liquid medium comprises the following components in final concentrations: GIBCO serum replacement: GIBCO serum replacement: 10 wt%; EW-7197-HCl: 1000 nM; SB 203580-HCl: 20 mu M; y-27632 dihydrochloride: 25 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; the solvent is DMEM/F-12 medium.
And 7, replacing the liquid culture medium every 2-3 days, and culturing for 7 days to obtain the lung cancer organoid with good activity.
Example 3
The present embodiment provides a method for feeding umbilical cord mesenchymal stem cells to an intestinal cancer organoid, which adopts the structural model of embodiment 1, and the method comprises the following steps:
step 1, preparing a feeding layer: inoculating human umbilical cord mesenchymal stem cells to a culture dish, culturing until the confluence degree of the cells is 60%, adding 4 microgram/ml of mitomycin C into a human umbilical cord mesenchymal stem cell culture medium, treating for 8 hours, and removing the mitomycin C to obtain a feeding layer;
step 2, tissue pretreatment: pretreating a fresh-source surgical excision specimen or biopsy tissue, wherein the pretreatment step comprises washing, chopping, digesting tissue blocks, filtering to remove impurities and the like to obtain a cell mass with the cell number of 3-50 cells, and centrifuging to remove a supernatant for later use;
step 3. mu.l of gel medium (containing Matrigel component) was added to the center of the inserted cell culture plate Hanging cell inserts membrane placed upside down in a sterilized plastic box matching the size of the cell culture dish, the box was closed, 5% CO was added at 37 ℃ C2Culturing for 15min in an incubator until the gel is solid for use; the gel medium included the following components at the final concentrations: GIBCO serum replacement: 5 wt%; EW-7197-HCl: 2000 nM; SB 203580-HCl: 40 mu M; y-27632 dihydrochloride: 2 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; matrigel: 3 mg/ml; the solvent is DMEM/F-12 culture medium;
step 4. resuspending 2. mu.l of the cell pellet obtained in step 2 in the gel medium (containing Matrigel component), mixing the cell pellet with a pipette, adding the cell pellet to the gel drop solidified by the Hanging cell inserts obtained in step 3 to form a gel drop, closing the box, and adding 5% CO at 37 ℃ to obtain a gel drop2Culturing for 20min in an incubator until the gel is solid for use;
step 5. mu.l of the above gel medium (containing Matrigel component) was added to the solidified double-layered gel drop obtained in step 4 to cover the previous gel drop, the box was closed, and 5% CO was added at 37 ℃ to2Culturing for 20min in an incubator until the gel is solid for use;
and 6, Hanging the Hanging cell inserts obtained in the step 5 on a culture dish inoculated with the human umbilical cord mesenchymal stem cell feeder layer prepared in the step 1 in a positive mode, and then adding a liquid culture medium into the culture dish to enable the glue drops on the Hanging cell inserts to be fully immersed in the liquid level of the culture medium. Then placing in a constant temperature incubator at 37 ℃ and 5% CO2At the concentration ofCulturing; the liquid medium comprises the following components in final concentrations: GIBCO serum replacement: 5 wt%; EW-7197-HCl: 2000 nM; SB 203580-HCl: 40 mu M; y-27632 dihydrochloride: 2 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; the solvent is DMEM/F-12 culture medium;
and 7, replacing the liquid culture medium every 2-3 days, and culturing for 7 days to obtain the intestinal cancer organoid with good activity.
Example 4
This example provides a method for feeding breast cancer organoids with umbilical cord mesenchymal stem cells, using the structural model of example 1, the method comprising the steps of:
step 1, preparing a feeding layer: inoculating human umbilical cord mesenchymal stem cells to a culture dish, culturing until the cell confluency is 40%, adding 10 microgram/ml mitomycin C into a human umbilical cord mesenchymal stem cell culture medium, treating for 4h, and removing the mitomycin C to obtain a feeding layer;
step 2, tissue pretreatment: pretreating a fresh-source surgical excision specimen or biopsy tissue, wherein the pretreatment step comprises washing, chopping, digesting tissue blocks, filtering to remove impurities and the like to obtain a cell mass with the cell number of 3-50 cells, and centrifuging to remove a supernatant for later use;
step 3. Add 4. mu.l of gel media (containing Matrigel component) to the center of the insert cell culture plate Hanging cell inserts membrane placed upside down in a sterilized plastic box matching the size of the cell culture dish, close the box, 5% CO at 37 deg.C2Culturing for 15min in an incubator until the gel is solid for use; the gel medium included the following components at the final concentrations: GIBCO serum replacement: 25 wt%; EW-7197-HCl: 100 nM; SB 203580-HCl: 1 mu M; y-27632 dihydrochloride: 50 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; matrigel: 6 mg/ml; the solvent is DMEM/F-12 culture medium;
step 4, taking 6 mul of the gel culture medium (containing Matrigel components) to re-suspend the cell mass sediment obtained in the step 2, evenly mixing the cell mass sediment by using a liquid transfer machine, adding the cell mass sediment to the gel drops solidified by the Hanging cell inserts obtained in the step 3, and forming a gelSmall drop, close box, 5% CO at 37 ℃2Culturing for 15min in an incubator until the gel is solid for use;
step 5. add 12. mu.l of the above gel medium (containing Matrigel component) to the solidified double-layered gel drop obtained in step 4 to cover the previous gel drop, close the box, and 5% CO at 37 deg.C2Culturing for 15min in an incubator until the gel is solid for use;
and 6, Hanging the Hanging cell inserts obtained in the step 5 on a culture dish inoculated with the human umbilical cord mesenchymal stem cell feeder layer prepared in the step 1 in a positive mode, and then adding a liquid culture medium into the culture dish to enable the glue drops on the Hanging cell inserts to be fully immersed in the liquid level of the culture medium. Then placing in a constant temperature incubator at 37 ℃ and 5% CO2Culturing under the concentration; the liquid medium comprises the following components in final concentrations: GIBCO serum replacement: 25 wt%; EW-7197-HCl: 100 nM; SB 203580-HCl: 1 mu M; y-27632 dihydrochloride: 50 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; the solvent is DMEM/F-12 culture medium;
and 7, replacing the liquid culture medium every 2-3 days, and culturing for 7 days to obtain the breast cancer organoid with good activity.
Example 5
In this example, the morphological identification and functional verification of the lung cancer organoids obtained in example 2 are specifically as follows:
1) organoid collection and fixation: the mixture was put into a prepared fixing solution (4% formaldehyde fixation) and fixed for 2 hours. Centrifuging at 1200rpm for 5 minutes after fixation is finished, and discarding formalin fixing solution;
2) gradient dehydration: immersing the fixed organoids in 85% alcohol, 95% alcohol and 100% alcohol for 30 minutes respectively;
3) transparent wax dipping: adding xylene to immerse the organoids for 20 minutes, and repeating twice; then adding paraffin wax, and soaking the paraffin wax for 1.5 hours at 60 ℃;
4) embedding the section: wrapping the organoid with an embedding mould, then slicing into sections of 4-6 μm with a slicer, and attaching the sections to an anti-falling glass slide;
5) baking slices: placing the glass slide on a glass slide frame, placing the glass slide on an oven, and baking water and paraffin on the glass slide for 30min at 65 ℃;
6) dewaxing: dewaxing with xylene three times for 10 minutes each; then dipping and washing the fabric with 100 percent alcohol for three times, 1 minute each time; finally, soaking and washing for 1 minute by running water;
7) h & E staining: staining with hematoxylin for 8min, washing with water for 1min, differentiating with 1% hydrochloric acid alcohol for 1-2 s, washing with flowing water for 30min, staining with 1% eosin for 1-2min, and washing with flowing water for 1 min;
8) fixing after dyeing: sequentially immersing in 95% alcohol and 100% alcohol, each reagent twice, each time for 2 minutes;
9) and (3) transparent and sealing: using dimethylbenzene for transparence for 2min, taking out, airing and sealing with neutral gum;
10) the tissue morphology structure was observed under a normal optical microscope, as shown in fig. 3, indicating that the obtained lung cancer organoids had good tissue morphology.
Example 6
In this example, morphological identification and functional verification of the intestinal cancer organoids obtained in example 3 were performed, and the specific procedure was performed with reference to example 5, and as shown in fig. 4, it was revealed that the obtained intestinal cancer organoids had good tissue morphology.
Example 7
In this example, morphological identification and functional verification are performed on the breast cancer organoids obtained in example 4, and the specific operation refers to example 5, as shown in fig. 5, which shows that the obtained breast cancer organoids have good tissue morphology.
Comparative example 1
The comparative example provides a method for feeding lung cancer organoids with irradiated human endometrial stem cells that have lost proliferative function, comprising the steps of:
1) preparing a feeding layer: inoculating human endometrium stem cells onto a culture dish, culturing until the confluence degree of the cells is 50%, placing the culture dish paved with the human endometrium stem cells and culture solution into an X-ray irradiator for irradiation, wherein the irradiation dose is 80KV, the irradiation time is 30min, and the whole 96-hole plate paved with human mesenchymal stem cells after irradiation is placed in a constant-temperature incubator at 37 ℃ for storage and is used as a feeding layer for later use;
2) the rest of the procedure was referred to example 2.
After 7 days of culture, as shown in FIG. 6, the cell state was poor and it was difficult to form a lung cancer organoid having good activity.
Comparative example 2
The comparative example provides a method for feeding human endometrial stem cells which lose the proliferation function through irradiation to an intestinal cancer organoid, which comprises the following steps:
1) preparing a feeding layer: inoculating human endometrium stem cells onto a culture dish, culturing until the confluence degree of the cells is 50%, placing the culture dish paved with the human endometrium stem cells and culture solution into an X-ray irradiator for irradiation, wherein the irradiation dose is 80KV, the irradiation time is 30min, and the whole 96-hole plate paved with human mesenchymal stem cells after irradiation is placed in a constant-temperature incubator at 37 ℃ for storage and is used as a feeding layer for later use;
2) the rest of the procedure was referred to example 3.
After 7 days of culture, as shown in FIG. 7, the cell state was poor and it was difficult to form a clear intestinal cancer organoid.
Comparative example 3
The present comparative example provides a method for feeding breast cancer organoids with irradiated human endometrial stem cells that have lost proliferative function, comprising the steps of:
1) preparing a feeding layer: inoculating human endometrium stem cells onto a culture dish, culturing until the cell confluency is 40-60%, placing the culture dish paved with the human endometrium stem cells and culture solution into an X-ray irradiator for irradiation, wherein the irradiation dose is 80KV, the irradiation time is 30min, and the whole 96-well plate paved with the human mesenchymal stem cells after irradiation is placed in a constant temperature incubator at 37 ℃ for storage and is used as a feeder layer for later use;
2) the rest of the procedure was referred to example 4.
After 7 days of culture, as shown in FIG. 8, the number of cells was small and the formation of breast cancer organoids was impossible.
Comparative example 4
This comparative example provides a method for feeding a lung cancer organoid with mesenchymal stem cells having no proliferation function, which is described in example 2, except that umbilical cord mesenchymal stem cells are replaced with human mesenchymal stem cells. After 7 days of culture, as shown in fig. 9, the cell status was poor and the success rate of forming lung cancer organoids by culture was low.
Comparative example 5
This comparative example provides a method for feeding mesenchymal stem cells having no proliferation function to an intestinal cancer organoid, in which umbilical cord mesenchymal stem cells were replaced with human mesenchymal stem cells, and the rest was conducted in example 3. After 7 days of culture, as shown in FIG. 10, the cell activity was poor and it was difficult to form normal-sized intestinal cancer organoids.
Comparative example 6
This comparative example provides a method for feeding a breast cancer organoid with mesenchymal stem cells having no proliferation function, which is described in example 4, except that umbilical cord mesenchymal stem cells are replaced with human mesenchymal stem cells. After 7 days of culture, as shown in FIG. 11, the number of cells was small and the formation of breast cancer organoids was not possible.
Comparative example 7
The comparative example provides a method for feeding lung cancer organoids with umbilical cord mesenchymal stem cells, which adopts a common culture mode (non-suspension culture), and the operation flow refers to example 2, and the only difference is that: in the comparative example, the lung cancer cell mass is not inoculated by using the Hanging cell inserts, but the lung cancer cell gel drops mixed with the gel culture medium are directly planted on the feeding layer obtained in the first step, namely the gel layer is directly contacted with the feeding layer.
After 7 days of culture, the cells were scattered and lung cancer organoids could not be formed, as shown in FIG. 12.
Comparative example 8
The present comparative example provides a method for feeding lung cancer organoids with umbilical cord mesenchymal stem cells, the operational procedure being as in example 2, except that: the gel medium was a commercially available conventional Matrigel, and the liquid medium was a commercially available DMEM/F-12 medium.
After 7 days of culture, as shown in FIG. 13, the number of cells was small and lung cancer organoids could not be formed.
In summary, the culture method of the invention is characterized as follows:
the components of the culture medium do not need to adopt bovine serum albumin (FBS) which is the most common component in cell culture, and other large protein molecules such as hormone and the like do not need to be added. The components are simpler, the cost is saved, and the risk of foreign virus pollution and pathogenic factor infection in the use of heterologous serum is reduced. And the inhibitors added into the culture medium are all water-soluble inhibitors, so that DMSO dissolution is not needed, the toxicity of DMSO on in-vitro cell culture is reduced while the effect is ensured.
Compared with the mesenchymal stem cells of the marrow, the human umbilical cord mesenchymal stem cells have the advantages of wide source, convenient material acquisition, relative purity, rich content, low immunogenicity and the like.
The human umbilical cord mesenchymal stem cells adopted by the invention are taken as feeder cells, can secrete various specific cell factors, can better simulate a complex in-vivo environment, and avoids the complexity of manual growth factor adding operation and the uncertainty of content, thereby being beneficial to the growth and proliferation of organoids, the maintenance of related functions and gene phenotypes.
The suspension type culture structure mode adopted by the invention can avoid the direct interaction of organoid and feeder layer cells to influence the physiological functions of organoid and feeder layer cells, and the cells are arranged in the two layers of gel, thus being more beneficial to the self-organization differentiation of small cell clusters.
The suspension culture method avoids the adherence of the organoid caused by the gravity action in the traditional glue dripping method, and is more beneficial to the later-stage tests such as passage, observation, detection and the like.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (3)

1. A method for feeding epithelial source organoids by umbilical cord mesenchymal stem cells is characterized in that: culturing is performed using a structural pattern in which an epithelial-derived tissue is located at the center of the structural pattern, and the epithelial-derived tissue is coated with a first gel layer, a second gel layer, and a third gel layer in this order from the inside to the outside, the third gel layer is coated with a liquid layer, and an umbilical cord mesenchymal stem cell feeder layer is located below the third gel layer in the liquid layer; the method comprises the following steps:
step 1, preparing an umbilical cord mesenchymal stem cell feeder layer;
step 2, processing the epithelial source tissue into a cell mass with the cell number of 3-50 cells;
step 3, taking three parts of gel culture medium, and suspending the cell mass obtained in the step 2 by the first part of gel culture medium to obtain cell culture solution; the second gel culture medium is solidified into drops, and the concrete mode is as follows: adding a second portion of gel medium to the center of the inserted cell culture plate, and placing the inserted cell culture plate in a sterile, closed, 37 deg.C, 5% CO2Culturing under inverted condition for 15-30min, and coagulating to obtain the final product; then adding the cell culture solution to the gel drops and further forming double-layer gel drops comprising a first gel layer and a second gel layer; adding a third gel culture medium to the double-layer gel drops to form a third gel layer; the gel medium is composed of the following components in final concentration: GIBCO serum replacement: 5-30 wt%; EW-7197-HCl: 100-; SB 203580-HCl: 1-40 mu M; y-27632 dihydrochloride: 2-50 mu M; penicillin: 100U/ml; streptomycin: 0.1 mg/ml; matrigel: 3-6mg/ml, and the solvent is DMEM/F-12 culture medium;
step 4, adding an umbilical cord mesenchymal stem cell feeder layer into a liquid culture medium, completely soaking the three layers of glue drops obtained in the step 3 into the liquid culture medium, enabling the three layers of glue drops to be positioned above the feeder layer, and culturing until organoids are formed; the liquid medium comprises the following components in final concentration: GIBCO serum replacement: 5-30 wt%; EW-7197-HCl: 100-; SB 203580-HCl: 1-40 mu M; y-27632 dihydrochloride: 2-50 mu M; penicillin: 100U/ml; streptomycin: 0.1mg/ml, and the solvent is DMEM/F-12 medium.
2. The method of feeding umbilical cord mesenchymal stem cells to an epithelial-derived organoid according to claim 1, wherein: the process for preparing the umbilical cord mesenchymal stem cell feeder layer in the step 1 comprises the following steps: inoculating human umbilical cord mesenchymal stem cells, culturing until the cell confluency is 40-60%, adding 4-10 μ g/ml mitomycin C, treating for 4-8h, and removing mitomycin C.
3. The method of feeding umbilical cord mesenchymal stem cells to an epithelial-derived organoid according to claim 1, wherein: the control conditions of culturing to organoid formation in the step 4 are as follows: at 37 ℃ with 5% CO2Culturing under the condition for 4-7 days, and replacing the liquid culture medium every 2-3 days.
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