CN114946837B - Tissue preservation solution for organoid culture and preparation method thereof - Google Patents
Tissue preservation solution for organoid culture and preparation method thereof Download PDFInfo
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- CN114946837B CN114946837B CN202210671714.XA CN202210671714A CN114946837B CN 114946837 B CN114946837 B CN 114946837B CN 202210671714 A CN202210671714 A CN 202210671714A CN 114946837 B CN114946837 B CN 114946837B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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Abstract
The invention discloses a tissue preservation solution for organoid culture, which comprises DMEM/F12 culture medium, penicillin-streptomycin, Y27632, HEPES and HGF mimics based on functional nucleic acid. The tissue preservation solution for organoid culture and the preparation method thereof are adopted, so that the survival and proliferation of cells are effectively stimulated, the cell activity is maintained, and the isolated tissue preservation and the subsequent organoid construction are facilitated.
Description
Technical Field
The invention relates to the technical field of isolated tissue preservation and transportation, in particular to tissue preservation solution for organoid culture and a preparation method thereof.
Background
Organoids (organoids) are 3D cell cultures cultured in vitro using adult stem cells, which have highly similar histological features to the corresponding human organ and reproduce part of the physiological functions of the organ. The isolated tumor tissue of a patient is utilized to construct organoid, and the generation process and the physiological and pathological states of the tumor tissue can be simulated, so that the method has wide application prospect in the aspects of basic research and clinical diagnosis and treatment.
The activity of the isolated tissue of the patient is the key to the subsequent culture of organoids. The existing tissue preservation method mainly comprises (1) transportation after ultralow temperature cryopreservation: the tissue is placed in liquid nitrogen or dry ice. (2) live cell transportation and preservation: placing living cells in culture solution, and storing and transporting at low temperature of 0-10 deg.C.
However, the transportation after cryopreservation has the problems of high cost, damage to cells in the cryopreservation-resuscitation process and the like, and is not beneficial to the maintenance of tissue activity and the construction of subsequent organoids. The existing living cell transportation and preservation methods are only suitable for short-time activity maintenance and long-time long-distance transportation, and have limited capability of maintaining the activity of tissues, thereby limiting the success rate of organoid construction.
Disclosure of Invention
The invention aims to provide a tissue preservation solution for organoid culture and a preparation method thereof, which can effectively stimulate the survival and proliferation of cells, thereby being beneficial to maintaining the cell activity of the cells and being beneficial to in vitro tissue preservation and subsequent organoid construction.
In order to achieve the above object, the present invention provides a tissue preservation solution for organoid culture, the preservation solution comprising DMEM/F12 medium, penicillin-streptomycin, Y27632, HEPES and an HGF mimic based on a functional nucleic acid.
Preferably, the mass concentration of penicillin-streptomycin is 0.9-1.2%, the molar concentration of Y27632 is 8-12 μ M, the molar concentration of HEPES is 9-13mM, and the molar concentration of HGF mimic is 0.9-1.3 μ M.
Preferably, the mass concentration of penicillin-streptomycin is 1%, the molar concentration of Y27632 is 10. Mu.M, the molar concentration of HEPES is 10mM, and the molar concentration of HGF mimic is 1. Mu.M.
Preferably, the HGF mimics based on the functional nucleic acids comprise functional nucleic acids Ap-1 and Ap-2, wherein Ap-1 is a nucleotide sequence shown as SEQ ID NO.1, and Ap-2 is a nucleotide sequence shown as SEQ ID NO. 2.
A preparation method of tissue preservation solution for organoid culture comprises the following steps:
s1, respectively preparing DMEM/F12 culture medium, Y-27632, HEPES, penicillin-streptomycin and HGF simulant solution according to concentration requirements;
s2, sequentially adding 10 mu M Y-27632, 10mM HEPES and 1% penicillin-streptomycin into a 20mL DMEM/F12 culture medium to prepare a preservation solution A;
and S3, adding 1 mu M of HGF simulant into the preservation solution A to obtain the preservative.
Preferably, the functional nucleic acid-based HGF mimics are prepared by the following method: respectively dissolving dry powders of functional nucleic acids Ap-1 and Ap-2 in pure water to prepare 100 mu M, respectively taking 12 mu L of mother liquor, adding 18 mu L of buffer solution, and fully and uniformly mixing to prepare 40 mu MAp-1 and Ap-2 solutions;
and (3) placing the prepared solution in a metal bath, heating at 95 ℃ for 5min, quickly placing on ice for half an hour, then mixing Ap-1 and Ap-2 with a ratio of 1.
Therefore, the tissue preservation solution for organoid culture and the preparation method thereof are beneficial to in vitro tissue preservation and subsequent organoid construction.
The invention uses functional nucleic acid to simulate the activity of HGF, adds the HGF into the preservation solution for preserving isolated tissues, and uses the capability of HGF to induce the survival and proliferation of cells, thereby improving the cell activity of the tissues preserved in the HGF, and being beneficial to long-time preservation and the construction of subsequent organoids.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a graphical representation of cell activities of mouse orthotopic prostate cancer tissues preserved in a preservation solution for 24h and 48h, respectively;
FIG. 2 is an image of isolated prostate cancer tissue under an optical microscope after culturing for 7 days after preserving in a preservation solution for 24h and 48h.
Detailed Description
The technical solution of the present invention is further illustrated by the accompanying drawings and examples.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art. These other embodiments are also covered by the scope of the present invention.
It should be understood that the above-mentioned embodiments are only for explaining the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent replacement or change of the technical solution and the inventive concept thereof in the technical scope of the present invention.
All terms (including technical or scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless specifically defined otherwise. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.
The disclosures of the prior art documents cited in the present description are incorporated by reference in their entirety and are therefore part of the present disclosure.
Example one
Synthesis of HGF mimetics based on functional nucleic acids
Dissolving dry powders of functional nucleic acids (Ap-1 and Ap-2) in merck water in a sterile operating platform to prepare 100 μ M, adding 18 μ L buffer solution into 12 μ L mother solution, and mixing to obtain 40 μ MAp-1 and Ap-2 solutions.
The 40 mu MAp-1 and Ap-2 solutions were heated separately in a metal bath at 95 ℃ for 5min and placed on ice quickly for half an hour. Then, ap-1 and Ap-2 are mixed with 1.
Example two
mu.M Y-27632, 10mM HEPES and 1% penicillin-streptomycin were sequentially added to 20mL of DMEM/F12 medium to prepare a preservation solution A, which was stored at 4 ℃. Additional 1 μ M HGF mimic was added prior to use.
Comparative example 1
10. Mu.M Y-27632, 10mM HEPES and 1% penicillin-streptomycin were sequentially added to 20mL of DMEM/F12 medium to prepare a storage solution A, and the mixture was mixed and dispensed into 5mL centrifuge tubes, each containing 2mL of the solution. The preservation solution was stored at 4 ℃.
Test of
1. Preservation of mouse prostate cancer tissue
Fresh C57bl/6 mice in situ prostate cancer tissue was cut into approximately 0.5X 0.5cm 3 The volume is measured, and the sample is put into a centrifuge tube filled with 2mL of preservation solution and is preserved in a refrigerator at 4 ℃. Respectively storing for 0h,24h and 48h. The preservation solutions prepared in comparative example one and example two were used as the preservation solutions, respectively. When the preservation time is up, the tissue is dissociated into single cell suspension, and the cell activity is detected by adopting a CCK8 cell activity detection kit according to the instruction.
As shown in FIG. 1, 1 and 2 in the figure are cell activity maps of 24h and 48h in the preservation solution of the first comparative example, 3 and 4 in the figure are cell activity maps of 24h and 48h in the preservation solution of the second example, and the cell activity maps are both statistical maps obtained by comparing with the cell activity of 0 h. According to the analysis of results, the cell activity of the preservation solution of the first comparative example and the cell activity of the preservation solution of the second example can be well maintained for 24 hours, and the cell activity of the preservation solution of the second example is higher than that of the preservation solution of the first comparative example. The preservation solution of the first comparative example can be preserved for 48 hours, while the cell activity in the preservation solution of the second example after 48 hours still reaches 80 percent, and the cell activity is greatly improved.
2. Culture of prostate cancer organoids in mice
Fresh C57bl/6 mice in situ prostate cancer tissue was cut into approximately 0.5X 0.5cm 3 The volume is measured, and the sample is put into a centrifuge tube filled with 2mL of preservation solution and is preserved in a refrigerator at 4 ℃. Respectively storing for 0h,24h and 48h. The preservative solutions were used separatelyThe preservation solutions of comparative example one and example two.
When the preservation time is up, organoid culture is carried out, and the specific steps are as follows: tissues were washed with pre-cooled PBS. The minced tissue mass was placed in a 15mL centrifuge tube, added with 1mL 2mg/mL Collagenase II (final concentration 1mg/mL containing 1% PS +10 μ MY-26732, diluted with DMEM/F12), digested for 1h at 37 ℃ with shaking at 90rpm, during which time the tube was gently tapped with a pipette every 15min to allow for full digestion. The Collagenase II was removed, 1ml of a preheated DMEM/F12 medium (DMEM/F12 +1% double antibody +10mM HEPES) was added to wash the tissue mass, the tissue mass was centrifuged at 250g for 5min at 4 ℃, the cells obtained were resuspended in the medium, and the cell mass was counted through a 100 μm cell filter.
All cell suspensions were centrifuged at 250g for 5min at 4 ℃ to adjust the cell density to 5000 cells/. Mu.L. About 80% final concentration of 12. Mu.L 5000 cells/. Mu.L + 48. Mu.L Martrigel was taken, 50. Mu.L of the cell-mixed Matrigel was dropped into a preheated 24-well plate, and 400. Mu.L of the culture solution was added after solidification for culture. The solution was changed every 3 days and observed 7 days later.
Isolated prostate cancer tissues stored in the storage solutions of comparative example one and example two for 24h and 48h were cultured for 7d and imaged using an optical microscope. As a result, it was found that the number of tissue-forming organoids stored in the storage solution 24h of example two was greater and the morphology was more beautiful than that of the storage solution 24h of comparative example one. The tissue preserved in the preservation solution of comparative example one for 48 hours failed to form a good organoid and cells were apoptotic. The tissue preserved in the preservation solution 48h of the second example can still well form organoids.
Therefore, the tissue preservation solution for organoid culture and the preparation method thereof are adopted, so that the survival and proliferation of cells are effectively stimulated, the cell activity is favorably maintained, and in-vitro tissue preservation and subsequent organoid construction are favorably realized.
Finally, it should be noted that: the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.
Sequence listing
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<120> tissue preservation solution for organoid culture and preparation method thereof
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Claims (4)
1. A tissue preservation solution for organoid culture, characterized in that: the components of the preservation solution are DMEM/F12 culture medium, penicillin-streptomycin, Y27632, HEPES and HGF mimics based on functional nucleic acid;
the mass concentration of penicillin-streptomycin is 0.9-1.2%, the molar concentration of Y27632 is 8-12 μ M, the molar concentration of HEPES is 9-13mM, and the molar concentration of HGF simulant is 0.9-1.3 μ M;
the HGF simulacrum based on the functional nucleic acid comprises functional nucleic acids Ap-1 and Ap-2, wherein Ap-1 is a nucleotide sequence shown as SEQ ID NO.1, and Ap-2 is a nucleotide sequence shown as SEQ ID NO. 2.
2. The tissue preservation solution for organoid culture according to claim 1, wherein: the mass concentration of penicillin-streptomycin was 1%, the molar concentration of Y27632 was 10. Mu.M, the molar concentration of HEPES was 10mM, and the molar concentration of HGF mimic was 1. Mu.M.
3. A method for producing the tissue preservation solution for organoid culture according to claim 1 or 2, characterized by comprising the steps of:
s1, respectively preparing DMEM/F12 culture medium, Y-27632, HEPES, penicillin-streptomycin and HGF simulant solution according to concentration requirements;
s2, sequentially adding 10 mu MY-27632, 10mMHEPES and 1% penicillin-streptomycin into a 20mLDMEM/F12 culture medium to prepare a preservation solution A;
and S3, adding 1 mu MHGF simulant into the preservation solution A to obtain the preservative solution.
4. The method for producing a tissue preservation solution for organoid culture according to claim 3, wherein the functional nucleic acid-based HGF mimetic is produced by the method comprising: respectively dissolving dry powders of functional nucleic acids Ap-1 and Ap-2 in pure water to prepare 100 mu M, respectively taking 12 mu L of mother liquor, adding 18 mu L of buffer solution, and fully and uniformly mixing to prepare 40 mu MAp-1 and Ap-2 solutions; and (3) placing the prepared solution in a metal bath, heating at 95 ℃ for 5min, quickly placing on ice for half an hour, then mixing Ap-1 and Ap-2 with a ratio of 1.
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| EP2412800A1 (en) * | 2010-07-29 | 2012-02-01 | Koninklijke Nederlandse Akademie van Wetenschappen | Liver organoid, uses thereof and culture method for obtaining them |
| US9051351B2 (en) * | 2011-04-02 | 2015-06-09 | Washington State University | Hepatocyte growth factor mimics as therapeutic agents |
| WO2016039371A1 (en) * | 2014-09-09 | 2016-03-17 | 国立大学法人東京大学 | Aptamer capable of binding to hgf receptor |
| CN112841168B (en) * | 2020-12-31 | 2021-12-10 | 创芯国际生物科技(广州)有限公司 | Tissue preservation solution and application thereof |
| CN112680398B (en) * | 2021-01-18 | 2023-03-17 | 南昌五元生物科技有限公司 | Culture medium for storing organoid at room temperature and method for maintaining growth activity of organoid |
| CN112690272B (en) * | 2021-01-18 | 2022-08-02 | 南昌五元生物科技有限公司 | Low-temperature preservation solution and preservation method thereof |
| CN113481162B (en) * | 2021-07-01 | 2023-02-24 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing tumor organoid |
| CN114391535B (en) * | 2022-01-19 | 2024-02-02 | 宋伟 | Sample preservation solution for culturing organoids |
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