CN114574431A - Method for culturing organoid or spheroid - Google Patents

Method for culturing organoid or spheroid Download PDF

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CN114574431A
CN114574431A CN202210336639.1A CN202210336639A CN114574431A CN 114574431 A CN114574431 A CN 114574431A CN 202210336639 A CN202210336639 A CN 202210336639A CN 114574431 A CN114574431 A CN 114574431A
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organoids
spheroids
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黄嘉钱
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Saibaisi Technology Co ltd
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Abstract

A method for culturing organoid or spheroids features that the hydrogel based on recombinant photosensitive protein is used as culture medium in which the organoid or spheroids can be wrapped for growing, and then analyzed or detected.

Description

Method for culturing organoid or spheroid
Technical Field
The invention belongs to the technical field of biological culture, and particularly relates to a culture method of organoid or spheroid.
Background
The study of mammalian organs and tissues has been a long-standing challenge because they are difficult to observe and analyze in real time. Alternatively, whole organs and organ sections have been routinely extracted and cultured in vitro. However, limited diffusion through these explants limits the use of this method to embryos or thin organs. Recently, significant advances in stem cell biology have shown that adult stem cells and pluripotent stem cells have the ability to survive, grow and differentiate in vitro into homeostatic tissue-mimicking structures or organoids when cultured in a three-dimensional matrix. This technological advance is becoming an important tool in understanding various biological processes occurring in vivo, such as tissue development and homeostasis, stem cell niche function, and tissue response to drugs, mutations, or injury. However, these cultures are still under development, which hinders their standard application in drug screening and therapy development. Three major limitations of the in vitro organoids or spheroids currently available;
the current culture conditions cannot simulate natural microenvironment, namely biomechanics, growth factors and signal gradients, which strongly limit the control of organoid growth, and the growth matrix represented by Matrigel is a commonly used cancer-derived matrix, which is often used as a scaffold for organoid or spheroid growth, and the organoid or spheroid is unevenly distributed on the factors of vitality, size, shape and the like, so that strong heterogeneity exists in the aspect of signal conduction, and the development of phenotypic analysis is hindered.
In particular, the culture substrate of organoids or spheroids needs to be greatly improved to meet the requirements of scientific research users or medical users on the characteristics of determination of the components of the culture substrate, easy operation and the like. At present, many studies are focused on the use of hydrogels of chemically synthesized polymer-based materials for organoid or spheroid culture, however, these synthetic materials have difficulty gaining wide acceptance in the market as compared to conventional animal-derived protein glues. In addition, however, batch-to-batch variation in matrigel can lead to inconsistent cell behavior, thereby introducing unknown and potential confounding variables that complicate the interpretation of basic and transformation studies. In addition, while matrigel is a key element of current organoid culture models, its role in organoid formation has not been elucidated. Therefore, there is a need to develop a new method sufficient to replace the above method in the organoid and spheroid fields.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a culture method of organoids or spheroids, which adopts hydrogel based on recombinant photosensitive protein as a culture substrate, and the organoids or spheroids can be wrapped in the culture substrate for growth and further used for analysis and detection.
In order to achieve the purpose, the invention adopts the technical scheme that:
a culture method for culturing organoids or spheroids, comprising the steps of: adding 10% Gibco FBS and 1% penicillin-streptomycin mixed solution into the culture medium, mixing the culture medium solution with organoids or spheroids, allowing the culture medium solution to form solid gel by itself or under the action of an auxiliary factor, and allowing the gel to contact with a liquid culture medium for culture.
Further, the culture may be released from the solid gel under light conditions.
Further, it comprises a basal medium, a recombinant light-sensitive protein (0.01-200 mg/mL), a cofactor 0.01-100 mM, gelatin 0.01-200mg/mL, collagen 0.01-200mg/mL, laminin 0.01-200. mu.g/mL, entactin 0.01-200. mu.g/mL, or a combination thereof.
Further, the recombinant photosensitive protein is a mixture of one or more recombinant photosensitive proteins, namely a recombinant protein component 1 and a recombinant protein component 2, freeze-dried powder of the recombinant protein component 1 and the recombinant protein component 2 are respectively dissolved into solutions with the weight fraction of 5% by using PBS, and the two solutions are mixed and added with 10mM adenine cobalamin aqueous solution under the condition of low light to generate the recombinant photosensitive protein hydrogel.
Further, the light-sensitive protein has a cofactor binding domain selected from a cobalamin binding domain, a flavin binding domain, a carotene binding domain, a chlorophyll binding domain, or a lutein binding domain.
Furthermore, the hydrogel can additionally contain gelatin, collagen, laminin or entactin, and the mechanical properties of the hydrogel can be regulated by light.
Further, the spheroid of the organoid is selected from a liver cell spheroid, a pancreas beta cell spheroid, a cardiac muscle cell spheroid, a glial cell spheroid, a skin epithelial cell spheroid, a cartilage cell spheroid, a bone cell spheroid, a breast cell spheroid, a lung cell spheroid, a kidney cell spheroid, and a muscle cell spheroid.
Further, the basic culture medium is a DMEM culture medium, an RPMI culture medium and an RPMI culture medium.
Further, organoid culture includes the following steps:
(1) encapsulating primary cells or cell aggregates isolated from an organ or differentiated to some extent from stem cells into a hydrogel;
(2) the hydrogel is placed under appropriate conditions for culturing.
The organoids are selected from the group consisting of intestinal organoids, retinal organoids, kidney organoids, liver organoids, stomach organoids, prostate organoids, breast organoids, inner ear organoids, cardiac fiber organoids, liver endothelial organoids, pancreas organoids, fallopian tube organoids, and brain organoids.
Compared with the prior art, the invention has the advantages that:
compared with the prior art, the hydrogel matrix is derived from recombinant protein, so that the problems of poor batch stability and poor controllability of material properties of animal-derived materials are solved.
Drawings
FIG. 1 is an embodiment of an MDA-MB-231 spheroid product of the invention;
FIG. 2 is a HCT116 spheroid embodying features of the invention;
FIG. 3 shows the intestinal organoid culture (10 days) of mice in the examples of the present invention.
Detailed Description
The invention will be further described with reference to the following drawings, but the invention is not limited to the following examples:
example 1
Preparing a recombinant photosensitive protein hydrogel;
the recombinant photosensitive protein is a mixture of one or more recombinant photosensitive proteins, preferably, the recombinant photosensitive proteins are ACA and BCB, which contain a cobalamin binding domain, and the amino acid sequence of the recombinant photosensitive protein is shown in figure X. Dissolving ACA and BCB lyophilized powder with PBS respectively to obtain 5% weight fraction solution, mixing the two solutions under low light condition, and adding appropriate amount of 10mM adenine cobalamin water solution to obtain colloidal solid.
Example 2
Culturing MDA-MB-231 spheroids;
after thawing the cells from liquid nitrogen, the cells were stored in DMEM medium in a cell culture flask, to which 10% Gibco FBS and 1% penicillin-streptomycin mixed solution were added. Subculture followed the ATCC protocol, with DMEM media formula as follows: 265.00 mg/L of calcium chloride dihydrate, 42.00 mg/L of L-serine, 0.10 mg/L of ferric nitrate nonahydrate, 95.00 mg/L of L-threonine, 400.00 mg/L of potassium chloride, 16.00 mg/L of L-tryptophan, 97.67 mg/L of anhydrous magnesium sulfate, 72.00 mg/L of L-tyrosine, 6400.00 mg/L of sodium chloride, 94.00 mg/L of L-valine, 109.00 mg/L of anhydrous sodium dihydrogen phosphate, 4.00 mg/L of D-calcium pantothenate, 75.00 mg/L of succinic acid, 7.20 mg/L of choline tartrate, 100.00 mg/L of sodium succinate, 4.00 mg/L of folic acid, 84.00 mg/L-arginine hydrochloride, 7.20 mg/L inositol, 63.00 mg/L-cystine hydrochloride, 4.00 mg/L nicotinamide, 30.00 mg/L glycine, 0.40 riboflavin, 42.00 mg/L-histidine hydrochloride, 4.00 mg/L thiamine hydrochloride, 105.00 mg/L-isoleucine, 4.00 mg/L pyridoxine hydrochloride, 105.00 mg/L-leucine, 1000.00 mg/L glucose, 146.00 mg/L-lysine hydrochloride, 110.00 mg/L sodium pyruvate, 30.00 mg/L-methionine, 9.30.00 mg/L phenol red, 66.00 mg/L-phenylalanine.
On the day of the experiment, the medium was aspirated from the flask, the cells were washed once in PBS buffer and dissociated using 1-1.5 ml of trypsin reagent. The trypsin reagent was neutralized using 4 times the volume of complete medium and the viable cell count and viability were captured using a cell count chamber.
Cells with > 90% survival were taken for spheroid generation. The cell stock was diluted 1:10 to 1:20 in complete medium to allow easier calculation of cell seeding density. The number of seeded cells was calculated using a cell seeding calculator. The desired number of cells was seeded into each well of the U-shaped spheroid well plate using a multichannel pipettor. The final volume was kept at 100. mu.l. Plates were centrifuged at 290 g for 3 minutes and placed in an incubator. This is day 0.
On day 1, 50. mu.L of 5% ACA, 50. mu.L of 5% BCB, and 10. mu.L of 10mM aqueous solution of adenine cobalamin were added to each well, and after careful mixing, the plate was centrifuged at 100 g for 3 minutes and then returned to the incubator.
Depending on the number of cells seeded, the spheres were completed in culture from day 4 (> 2,000 cells) to day 7 (< 2,000 cells).
Example 3
Culture of LNCaP spheroids
After thawing the cells from liquid nitrogen, the cells were stored in RPMI medium in a cell culture flask, to which 10% Gibco FBS and 1% penicillin-streptomycin mixed solution were added. Subculture following ATCC protocol, RPMI media composition: 10.0mg/L of glycine, 200.0mg/L of L-arginine, 50.0mg/L of L-asparagine, 20.0mg/L of L-aspartic acid, 65.0mg/L of L-cystine 2HCl, 20.0mg/L of L-glutamic acid, 300.0mg/L of L-glutamine, 15.0mg/L of L-histidine, 20.0mg/L of L-hydroxyproline, 50.0mg/L of L-isoleucine, 50.0mg/L of L-leucine, 40.0mg/L of L-lysine hydrochloride, 15.0mg/L of L-methionine, 15.0mg/L of L-phenylalanine, 20.0mg/L of L-proline, 30.0mg/L of L-serine, 20.0mg/L of L-threonine, l-tryptophan 5.0mg/L, L-disodium tyrosine dihydrate 29.0mg/L, L-valine 20.0mg/L, biotin 0.2mg/L, choline chloride 3.0mg/L, D-calcium pantothenate 0.25mg/L, folic acid 1.0mg/L, nicotinamide 1.0mg/L, p-aminobenzoic acid 1.0mg/L, pyridoxine hydrochloride 1.0mg/L, riboflavin 0.2mg/L, thiamine hydrochloride 1.0mg/L, vitamin B120.005mg/L, i-inositol 35.0mg/L, calcium nitrate (Ca (NO 3)) 24H 2O) 100.0mg/L, magnesium sulfate (MgSO4) (anhydrous) 48.84mg/L, potassium chloride (KCl) 400.0mg/L, sodium bicarbonate (NaHCO3) 2000.0mg/L, sodium chloride (0.0 mg)/L, 800.0mg/L of anhydrous disodium hydrogen phosphate (Na2HPO4), 2000.0mg/L of D-glucose (glucose), 1.0mg/L of glutathione (reduced state) and 5.0mg/L of phenol red.
Once the cells reached 70-80% coverage in the flask, the medium was aspirated from the flask, the cells were washed once in 1X PBS, and dissociated using 1-1.5 ml of trypsin reagent. The trypsin reagent was neutralized using 4 times the volume of complete medium and the viable cell count and viability were captured using a cell count chamber. Cells with > 90% survival were taken for spheroid generation.
The cell stock was diluted 1:10 to 1:20 in complete medium to allow easier calculation of cell seeding density. The number of seeded cells was calculated using a cell seeding calculator.
On day 0, the required number of cells was seeded into each well of the U-shaped spheroid well plate using a multichannel pipette. The final volume was kept at 200. mu.l. The plates were centrifuged at 1,500 rpm for 10 minutes and then placed in an incubator at 37 ℃ and 5% CO 2.
On the first day, 50. mu.L of 5% ACA, 50. mu.L of 5% BCB, and 10. mu.L of 10mM aqueous solution of adenosylcobalamin were added to each well, and after careful mixing, the plate was centrifuged at 100 g for 3 minutes and then returned to the incubator. The 1:1 medium was changed every other day (100. mu.l of used medium was aspirated and 100. mu.l of fresh medium was added). After medium exchange, the plate was centrifuged at 1,200 rpm for 5 minutes and returned to the incubator having the above conditions. On days 4-8, the spheres had been cultured.
Example 4
Culture of HCT116 spheroids
After thawing the cells from liquid nitrogen, the cells were stored in RPMI medium in a cell culture flask, to which 10% Gibco FBS and 1% penicillin-streptomycin mixed solution were added. Subculture following ATCC protocol, RPMI media composition: 10.0mg/L of glycine, 200.0mg/L of L-arginine, 50.0mg/L of L-asparagine, 20.0mg/L of L-aspartic acid, 65.0mg/L of L-cystine 2HCl, 20.0mg/L of L-glutamic acid, 300.0mg/L of L-glutamine, 15.0mg/L of L-histidine, 20.0mg/L of L-hydroxyproline, 50.0mg/L of L-isoleucine, 50.0mg/L of L-leucine, 40.0mg/L of L-lysine hydrochloride, 15.0mg/L of L-methionine, 15.0mg/L of L-phenylalanine, 20.0mg/L of L-proline, 30.0mg/L of L-serine, 20.0mg/L of L-threonine, l-tryptophan 5.0mg/L, disodium L-tyrosine dihydrate 29.0mg/L, L-valine 20.0mg/L, biotin 0.2mg/L, choline chloride 3.0mg/L, calcium D-pantothenate 0.25mg/L, folic acid 1.0mg/L, nicotinamide 1.0mg/L, p-aminobenzoic acid 1.0mg/L, pyridoxine hydrochloride 1.0mg/L, riboflavin 0.2mg/L, thiamine hydrochloride 1.0mg/L, vitamin B120.005mg/L, i-inositol 35.0mg/L, calcium nitrate (Ca (NO 3)) 24H 2O) 100.0mg/L, magnesium sulfate (MgSO4) (anhydrous) 48.84mg/L, potassium chloride (KCl) 400.0mg/L, sodium bicarbonate (NaHCO3) 2000.0mg/L, sodium chloride (0.0 mg/L), 800.0mg/L of anhydrous disodium hydrogen phosphate (Na2HPO4), 2000.0mg/L of D-glucose (glucose), 1.0mg/L of glutathione (reduced state) and 5.0mg/L of phenol red.
Once the cells reached 70-80% coverage in the flask, the medium was aspirated from the flask, the cells were washed once in 1X PBS, and dissociated using 1-1.5 ml of trypsin reagent. The trypsin reagent was neutralized using 4 times the volume of complete medium and the viable cell count and viability were captured using a cell count chamber. Cells with > 90% survival were taken for spheroid generation.
The cell stock was diluted 1:10 to 1:20 in complete medium to allow easier calculation of cell seeding density. The number of seeded cells was calculated using a cell seeding calculator.
On day 0, the required number of cells was seeded into each well of the U-shaped spheroid well plate using a multichannel pipette. The final volume was kept at 200. mu.l. The plates were centrifuged at 1,500 rpm for 10 minutes and then placed in an incubator at 37 ℃ and 5% CO 2.
On the first day, 50. mu.L of 5% ACA, 50. mu.L of 5% BCB, and 10. mu.L of 10mM aqueous solution of adenosylcobalamin were added to each well, and after careful mixing, the plate was centrifuged at 100 g for 3 minutes and then returned to the incubator. The 1:1 medium was changed every other day (100. mu.l of used medium was aspirated and 100. mu.l of fresh medium was added). After medium exchange, the plate was centrifuged at 1,200 rpm for 5 minutes and returned to the incubator having the above conditions. On days 4-8, the spheres had been cultured.
Example 5
Culture of mouse intestinal organoid
First, after the mice were sacrificed by cervical dislocation, an intestinal section of about 15 cm from the terminal ileum was collected, placed in a pre-cooled D-PBS + antibiotic (1X) wash solution (containing antibiotics), the membrane, blood vessels and fat outside the intestinal tract were removed using a sterile sharp-tipped forceps, and the pre-cooled D-PBS + antibiotic (1X) was injected from one end of the small intestine using a syringe for rinsing. Using small scissors, the intestinal section was cut longitudinally with the intestinal lumen open upward. The dissected intestinal sections were gently washed with pre-cooled D-PBS + antibiotic (1X) (2-8 ℃), the intestinal contents and villous structures scraped off with a sterile slide, and rinsed again with pre-cooled D-PBS + antibiotic (1X). 15 mL of pre-cooled D-PBS + antibiotic (1X) was added to a 50 mL centrifuge tube. One end of the intestine is held by forceps and suspended over the orifice. Starting from the bottom of the intestine, the intestine was cut into 1-2 mm pieces with sterile scissors, and the pieces were allowed to fall into the buffer in the tube. Pre-wetting a 10 mL pipette with D-PBS + antibiotic (1X), and washing the intestinal debris 5-10 times by adding 15 mL of pre-cooled D-PBS + antibiotic (1X) until the supernatant becomes clear.
(II) remove the supernatant, resuspend the tissue fragments in 25 mL small intestine crypt digest (2-5 mM EDTA in D-PBS + antibiotic (1X) buffer), incubate on ice for 20 minutes, and rotate the ice box on a 20 rpm shaker for 30 minutes. The tissue fragments were allowed to settle naturally by gravity for about 1 minute. The digestive juices were carefully aspirated and discarded, leaving enough liquid to submerge the tissue fragments. The tissue fragments were resuspended in 10 mL of pre-cooled 10% FBS-containing D-PBS + antibiotic (1X) buffer and pipetted up and down three times. Rest until most of the intestinal tissue fragments settle to the bottom. The supernatant was carefully pipetted and filtered through a 70 μm or 100 μm sieve and the filtrate collected in a clean 50 mL centrifuge tube. Repeating the above steps 7-8 at least 2 times, and mixing the obtained three (or even more) filtrates.
(III) at 2-8 ℃ the mixture in 290 x g centrifugal 5 minutes (horizontal centrifugal rotor, not fixed angle rotor). Carefully pour out and discard the supernatant. The pellet was resuspended in 10 mL of D-PBS buffer. Centrifuge at 200 Xg for 3 min. The supernatant was decanted. The intestinal crypt pellet was left in the tube. 2 mL of basal medium (Advanced DMEM/F-12+10 mM Hepes +2 mM L-glutamine) was added to resuspend the pellet, and if there were too many single cells, the supernatant was centrifuged at 200 Xg for 2 minutes to remove the single cells. Using a pre-wetted pipette tip, 10. mu.L of the pipette is pipetted onto a hemocytometer. Using an inverted microscope, the number of crypts in a 10. mu.L sample was counted. No single cells or large multi-layered tissue fragments are counted. The volume of the selected fraction containing approximately 750, 1500, 3000 crypts was calculated. The desired volumes were transferred to three correspondingly labeled 15 mL conical tubes and centrifuged at 200 x g and 2-8 ℃ for 5 minutes. Carefully aspirate and discard the supernatant. The desired volumes were transferred to three correspondingly labeled 15 mL centrifuge tubes and centrifuged at 200 x g and 2-8 ℃ for 5 minutes. Carefully aspirate and discard the supernatant. mu.L of complete intestinal organ growth medium at room temperature (15-25 ℃) was added to each centrifuge tube. Cold medium is not used.
(IV) Add 150. mu.L of mixed culture medium (50. mu.L of Matrigel, 50. mu.L of 5% ACA, 50. mu.L of 5% BCB, and 10. mu.L of 10mM aqueous solution of adenosylcobalamin) to the centrifuge tube in the above step, carefully stir up and down several times to mix well, taking care to avoid air bubbles. After culturing for 7-10 days, the small intestine organoids can form a complex budding structure.

Claims (11)

1. A culture method for culturing organoids or spheroids, comprising the steps of: preparing the recombinant protein into a culture medium solution, mixing the culture medium solution with organoids or spheroids, allowing the culture medium solution to form solid gel by itself or under the action of a cofactor, and allowing the gel to contact with a liquid culture medium for culture.
2. The method of claim 1, wherein the culture is released from the solid gel under light conditions.
3. A culture medium for organoids or spheroids, comprising a basal medium, recombinant light-sensitive protein (0.01-200 mg/mL), and cofactor (0.01-100 mM).
4. The culture medium of an organoid or spheroid according to claim 3, further comprising gelatin 0.01-200mg/mL, collagen 0.01-200mg/mL, laminin 0.01-200 μ g/mL, or entactin 0.01-200 μ g/mL, or a combination thereof.
5. The organoid or spheroid culture medium of claim 3, wherein the recombinant light-sensitive protein is a mixture of one or more recombinant light-sensitive proteins, and is recombinant protein fraction 1 and recombinant protein fraction 2, wherein lyophilized powders of recombinant protein fraction 1 and recombinant protein fraction 2 are dissolved in 5% by weight of PBS, respectively, and the two solutions are mixed and added with an adenine cobalamin aqueous solution (0.1-100 mM) under the conditions of no light, low light or only red light to form the recombinant light-sensitive protein hydrogel.
6. The organoid or spheroid culture medium of claim 3, wherein the recombinant light sensitive protein has a cofactor binding domain selected from a cobalamin binding domain, a flavin binding domain, a carotene binding domain, a chlorophyll binding domain, or a lutein binding domain, and the corresponding cofactor is cobalamin, flavin, carotene, chlorophyll, lutein.
7. The organoid or spheroid culture medium of claim 4, wherein the hydrogel additionally comprises gelatin, collagen, laminin or entactin, and the mechanical properties of the hydrogel can be controlled by light.
8. The method of claim 1, wherein the organoid is selected from the group consisting of hepatocyte spheroids, pancreatic beta cell spheroids, cardiomyocyte spheroids, glial cell spheroids, dermal epithelial cell spheroids, chondrocyte spheroids, osteocyte spheroids, mammary cell spheroids, lung cell spheroids, renal cell spheroids, and muscle cell spheroids.
9. The method of claim 1, wherein the basal medium is DMEM medium, RPMI medium, or RPMI medium.
10. The method of claim 1, wherein the organoid culture comprises the steps of:
(1) encapsulating primary cells or cell aggregates isolated from an organ or differentiated to some extent from stem cells into a hydrogel;
(2) the hydrogel is placed under appropriate conditions for culturing.
11. The method of claim 1, wherein the organoids are selected from the group consisting of intestinal organoids, retinal organoids, kidney organoids, liver organoids, stomach organoids, prostate organoids, breast organoids, inner ear organoids, cardiac muscle fiber organoids, liver endothelial organoids, pancreas organoids, fallopian tube organoids, and brain organoids.
CN202210336639.1A 2022-04-01 2022-04-01 Method for culturing organoid or spheroid Withdrawn CN114574431A (en)

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