Cell preservation

Abstract

Provided is a technology for obtaining a cell having higher adhesiveness to a substrate. This cell is obtained by preserving a cell suspension, wherein the preservation of the cell suspension includes preserving in a temperature zone exceeding a refrigeration preservation temperature and less than a culture temperature.

Description

Cell preservation

[0001] The present invention relates to cell preservation and the like.

[0002] Groups of odorants that characterize specific human diseases and mental states have been identified, and because of their high utility as test markers, the development of various odor sensors targeting these has become active. Because biological olfactory receptors have superior properties in terms of diversity, sensitivity, selectivity, etc. that are not found in conventional odor sensor elements such as semiconductors, there are high expectations for the development of new odor sensors that use olfactory receptors as sensor elements.

[0003] Patent Document 1 discloses the use of cells expressing modified olfactory receptors or lipid bilayer membranes comprising modified olfactory receptors as odor sensors.

[0004] International Publication No. 2022 / 024902

[0005] Odor sensors, which involve the artificial preparation of lipid bilayer membranes containing sensor proteins such as olfactory receptors, are not necessarily manufactured efficiently, and further improvements in the manufacturing efficiency of odor sensors are needed. Therefore, we focused on using cells that express sensor proteins.

[0006] When cells are used as chemical sensors, such as odor sensors, from the viewpoint of ease of use, it is desirable to prepare cells in advance in a container or the like and use them when needed, rather than culturing and preparing the cells each time. In the latter case, from the viewpoints of large-scale production and production efficiency, it is desirable to temporarily store the cultured cells and prepare a large-scale stock of them and / or transport them to a more appropriate production site and seed them into a container, such as a culture plate, with compartments. From the viewpoint of signal detectability from the cells, it is preferable to form a cell layer on the substrate of the container in which the cells are seeded (e.g., the bottom surface of the compartments). From the viewpoints of production efficiency and signal detectability, it is desirable to exchange the medium after cell layer formation with a solution suitable for signal detection. During research, the inventors observed the state of the cell layer after this solution exchange and discovered a problem in which the cell layer detached from the substrate due to the solution exchange. This problem leads to differences in the state (degree of detachment) of the cells between cell samples (e.g., between wells), which is undesirable from the viewpoint of product uniformity.

[0007] Therefore, an objective of the present disclosure is to provide a technique for obtaining cells with higher adhesiveness to a substrate.

[0008] In the course of intensive research, the present inventors discovered that the storage temperature of cultured cells affects their adhesion to a substrate. Based on this finding, the present inventors conducted further research and found that the above-mentioned problems can be solved by cells obtained by storing a cell suspension, the storage of which includes storing the cell suspension at a temperature range above the refrigerated storage temperature and below the culture temperature. That is, the present disclosure encompasses the following aspects.

[0009] Item 1. Cells obtained by storing a cell suspension, wherein the storage of the cell suspension comprises storing the cell suspension in a temperature range above a refrigerated storage temperature and below a culture temperature.

[0010] Item 2. The cell according to Item 1, which is an insect cell.

[0011] Item 3. The cell according to Item 2, wherein the temperature range is 6 to 23°C.

[0012] Item 4. The cells according to any one of Items 1 to 3, wherein the storage time in the aforementioned temperature range is 12 hours or longer.

[0013] Item 5. The cells according to any one of Items 1 to 4, wherein the total storage time of the cell suspension is 12 hours or more.

[0014] Item 6. The cells according to any one of Items 1 to 5, wherein the total storage time of the cell suspension is 400 hours or less.

[0015] Item 7. The cells according to any one of Items 1 to 6, wherein the cell suspension is stored in a non-cell-adherent container.

[0016] Item 8. A cell chip comprising the cells according to any one of Items 1 to 7.

[0017] Item 9. A method for preparing cells, comprising storing a cell suspension, wherein the cell suspension is stored in a temperature range above refrigerated storage temperature and below culture temperature.

[0018] Item 10. A method for producing a cell chip, comprising seeding the cells according to any one of Items 1 to 7 to form a cell layer on a substrate.

[0019] According to the present disclosure, a technique for obtaining cells with higher adhesiveness to a substrate can be provided.

[0020] Photographs showing the results of the cell preservation test in Test Example 2 are shown. The storage conditions are as follows: A: 4°C (storage for 1 or 7 days), B: 10°C (storage for 1 or 7 days), C: 15°C (storage for 1 or 7 days), D: storage at 20°C for 1 day, or storage at 20°C for 1 day followed by storage at 4°C for 6 days. The total storage period is indicated on the left side of the photograph. Photographs showing the results of the cell preservation test in Test Example 3 are shown. The storage conditions are as follows: A: 4°C (storage for 1, 7, or 9 days), B: 10°C (storage for 1, 7, or 9 days), C: storage at 15°C for 1 day, or storage at 15°C for 1 day followed by storage at 10°C for 6 or 8 days, D: storage at 20°C for 1 day, or storage at 20°C for 1 day followed by storage at 10°C for 6 or 8 days. The total storage period is indicated on the left side of the photograph. Photographs showing the results of the cell preservation test in Test Example 4 are shown. The storage conditions are as follows: A: Stored at 4°C for 6 days, B: Stored at 10°C for 6 days, C: Stored at 12°C for 6 days, D: Stored at 14°C for 6 days, E: Stored at 16°C for 6 days. Photographs showing the results of the cell preservation test in Test Example 5 are shown. The storage conditions are as follows: A: Stored at 4°C for 1 day, B: Stored at 4°C for 7 days, C: Stored at 4°C for 1 day followed by 10°C for 6 days, D: Stored at 4°C for 8 days, E: Stored at 4°C for 1 day followed by 10°C for 7 days. The cell lines used are shown at the top of the photographs. #1 to #8 represent SpIm cell lines stably expressing olfactory receptors into which different olfactory receptors have been introduced.

[0021] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0022] In this specification, any combination of upper and lower limits of a certain numerical range is also directly and unambiguously disclosed.

[0023] In this specification, when a range is indicated by "equal to or greater than" and / or "equal to or less than," "equal to or greater than" and / or "equal to or less than" can be arbitrarily replaced with "greater than" and / or "less than."

[0024] In one aspect, the present disclosure relates to cells (sometimes referred to herein as "cells of the present disclosure") obtained by storing a cell suspension, the storage of which includes storing the cell suspension in a temperature range above refrigerated storage temperature and below culture temperature (the temperature range of the present disclosure). This will be described below.

[0025] The cells are not particularly limited. From the viewpoint of suitability for detecting chemical substances, animal cells such as insect cells and mammalian cells are preferred. Among them, insect cells are preferred from the viewpoint of ease of obtaining cells with higher adhesiveness to a substrate when stored in the temperature range of the present disclosure.

[0026] Examples of insect cells include Sf cells, MG1 cells, and High Five cells. TM Examples of Sf cells that can be used include Sf9 cells (ATCC CRL1711) and Sf21 cells. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0027] The cells derived from an insect of the family Arctiidae are primary cultured cells or established cell lines of cells derived from an insect of the family Arctiidae that constitute the living body, and are not particularly limited as long as they are so.

[0028] Examples of the Arctiidae family include the Arctiinae subfamily, Lithosiinae subfamily, and Syntominae subfamily, with the Arctiinae being preferred among these. Examples of the Arctiinae subfamily include the genera Spilosoma, Spilarctia, and Rhagonis, with the Rhagonis genus being particularly preferred. The Rhagonis genus is not particularly limited, but the Rhagonis genus is particularly preferred.

[0029] Cells derived from an insect of the family Arctiidae can be obtained from a known biobank, or can be obtained by collecting and culturing from a living insect of the family Arctiidae according to or in accordance with a known method, or by establishing a line if necessary.

[0030] Examples of cells derived from Mulberry butterfly include FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the National Agriculture and Food Research Organization Genebank.

[0031] A cell suspension contains cells and a solution to suspend the cells in. The solution is not particularly limited as long as it allows cells to be preserved, and is usually a solution containing water and with an appropriately adjusted osmotic pressure. The solution is preferably a culture medium.

[0032] The medium contains a carbon source and inorganic salts that can be utilized by the cells, and either a natural medium or a synthetic medium may be used.

[0033] Any carbon source that can be utilized by the cells may be used, for example, carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

[0034] Examples of nitrogen sources that can be used include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and digests thereof.

[0035] Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0036] More specific examples of media for insect cells include TNM-FH medium (Pharmingen), Sf-900 III SFM medium (Life Technologies), ExCell400, ExCell405 (both JRH Biosciences), and Grace's Insect Medium (Nature, 195, 788 (1962)).

[0037] The pH of the medium is, for example, 6 to 7.

[0038] In the cell suspension, the cells may be in a state where a single cell is separated from other cells, or in a state where multiple cells (e.g., 2 to 100, 2 to 50, 2 to 10, 2 to 5) form clumps of cells.

[0039] For preservation of the cell suspension, cells after culture can be used.

[0040] Cell suspensions are typically stored in a container. The container is not particularly limited. From the viewpoint of ease of cell seeding after storage, the container used for storage is preferably a non-cell-adhesive container, and more preferably has a coating applied to the inner surface that prevents cell adhesion. Various coatings that prevent cell adhesion are known (for example, JP 2020-156411 A), and examples of such coatings include immobilizing water-soluble resins (specific examples include saponified polyvinyl acetate, polyvinylpyrrolidone, polyethylene glycol, etc.), hydrophilic polymers (specific examples include poly-2-hydroxyethyl methacrylate (poly-HEMA), phosphorylcholine group-containing polymer compounds, polyethylene glycol chain-containing polymer compounds, 2-methacryloyloxyethyl phosphorylcholine-containing polymers, etc.) on the surface of a substrate. A non-cell-adhesive vessel is a vessel for a purpose other than cell adhesion, and examples thereof include vessels that do not have a substrate surface (cell well bottom) for cell retention, and specific examples thereof include centrifuge tubes, centrifuge tube-type bioreactor tubes with 4 to 8 holes of 1 to 5 mm in diameter in the lid, etc. (e.g., CELLSTAR, CELLreactor, centrifuge tube, conical bottom, 15 ml, 50 ml, manufactured by Greiner Bio-One), etc.

[0041] The method for filling the container with the cell suspension is not particularly limited. From the viewpoint of improving cell adhesion, the filling rate of the container with the cell suspension (= (cell suspension volume / container volume) × 100) is preferably 2% or more, more preferably 4% or more, even more preferably 6% or more, even more preferably 8% or more, and particularly preferably 10% or more; and is preferably 80% or less, more preferably 60% or less, even more preferably 50% or less, even more preferably 40% or less, and particularly preferably 30% or less. From the viewpoint of improving cell adhesion, the distance from the liquid surface to the bottom of the container during storage is preferably 30 mm or less, more preferably 25 mm or less, even more preferably 20 mm or less, even more preferably 15 mm or less, and particularly preferably 10 mm or less.

[0042] The mode of preserving the cell suspension is not particularly limited, and examples thereof include leaving the cell suspension to stand, shaking, etc. In a preferred mode of the present disclosure, the cell suspension is preserved by leaving the cell suspension to stand.

[0043] Preservation of the cell suspension includes preserving it at a temperature range above the refrigeration temperature and below the culture temperature (the temperature range of the present disclosure). This allows cells to be obtained with higher adhesiveness to the substrate. It also prevents cell death due to excessive cell proliferation and prevents a decrease in performance (e.g., sensitivity) as a chemical sensor.

[0044] The refrigerated storage temperature is the storage temperature for the purpose of refrigerated storage of cells, and such temperature ranges are known to those skilled in the art. The "refrigerated storage temperature" in the above storage is, for example, 8°C or lower, preferably 6°C or lower, and more preferably 5°C or lower, and the lower limit temperature is, for example, 1°C, preferably 2°C, and more preferably 3°C.

[0045] The culture temperature is the temperature at which cells are cultured and varies depending on the type of cell. In the case of insect cells, the "culture temperature" during storage is, for example, 21°C or higher, preferably 22°C or higher, more preferably 23°C or higher, even more preferably 24°C or higher, and particularly preferably 25°C or higher, with the upper limit being, for example, 30°C, 28°C, or 27°C.

[0046] From the viewpoint of the degree of improvement in adhesion to the substrate, the temperature range of the present disclosure is preferably 5°C or higher, more preferably 6°C or higher, even more preferably 7°C or higher, even more preferably 8°C or higher, and particularly preferably 9°C or higher. Furthermore, when the optimal culture temperature (the temperature at which the cell proliferation rate is maximum) is X°C, the temperature range is preferably X-2°C or lower, more preferably X-3°C or lower, even more preferably X-4°C or lower, even more preferably X-5°C or lower, and particularly preferably X-6°C or lower. In the case of insect cells, the temperature range of the present disclosure is preferably 5 to 25°C, more preferably 6 to 24°C, even more preferably 7 to 23°C, even more preferably 8 to 22°C, and particularly preferably 9 to 21°C. In a preferred embodiment, the temperature range is 18°C ​​or lower, 16°C or lower, 15°C or lower, 14°C or lower, 13°C or lower, 12°C or lower, or 11°C or lower.

[0047] The storage time in the temperature range of the present disclosure is, for example, 8 hours or more, and is preferably longer, for example, 12 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 96 hours or more, 120 hours or more, 144 hours or more, or 168 hours or more, as long as the cell viability is not affected. The storage time in the temperature range of the present disclosure is, for example, 400 hours or less, 350 hours or less, 300 hours or less, or 250 hours or less.

[0048] Storage of the cell suspension can also include storage at temperatures other than those disclosed herein (e.g., refrigeration). The total storage time (total storage time) including the storage time at the temperature range disclosed herein and the storage time at another temperature range is not particularly limited as long as it does not affect cell viability, and can be, for example, 12 hours or more, 24 hours or more, 48 hours or more, 72 hours or more, 96 hours or more, 120 hours or more, 144 hours or more, or 168 hours or more, or, for example, 400 hours or less, 350 hours or less, 300 hours or less, or 250 hours or less. The longer the total storage time, the greater the difference in the degree of cell adhesion to the substrate between storage at the temperature range disclosed herein and storage without storage at the temperature range disclosed herein.

[0049] When the storage of the cell suspension includes storage at other temperature ranges, the timing of storage at the temperature range of the present disclosure is not particularly limited. The intended effect can be achieved by storing the cell suspension at the temperature range of the present disclosure at any time during the storage period (e.g., at the start, middle, or end of the storage period).

[0050] The cells of the present disclosure are cells obtained by preserving a cell suspension after preservation.

[0051] The cells of the present disclosure are preferably contained in a cell chip. In one aspect, the present disclosure relates to a cell chip comprising a compartment containing the cells of the present disclosure.

[0052] The container used in the cell chip of the present disclosure is not particularly limited as long as it is capable of retaining cells. From the viewpoint of signal detectability from the cells, it is preferable to form a cell layer on the substrate of the container in which the cells are seeded (e.g., the bottom surface of the compartment). From the viewpoint of production efficiency and signal detectability, it is desirable to replace the culture medium with a solution suitable for signal detection after the cell layer is formed. Therefore, it is preferable that the container used in the cell chip has a surface on which a cell layer can be formed and has a shape suitable for liquid exchange of multiple samples. For this reason, the cell chip of the present disclosure preferably has a cell layer containing the cells of the present disclosure disposed on a substrate within the cell chip, and preferably includes cells and compartments for retaining the cells.

[0053] A cell layer is a layer formed by the accumulation of cells. After seeding a cell suspension on a substrate, a cell layer can be formed on the substrate by utilizing a force (gravity, centrifugal force, etc.) toward the substrate.

[0054] The compartment can be, for example, a cell or a compartment within an apparatus for holding the cells (such as a dish or well plate).

[0055] The shape of the compartment is not particularly limited as long as it is capable of retaining cells. From the viewpoints of cell retention, production efficiency, etc., the compartment is preferably in the form of a well.

[0056] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0057] The area of ​​the compartment is preferably 0.5 to 100 mm from the viewpoint of detection sensitivity or production efficiency. 2 , more preferably 1 to 50 mm 2 , more preferably 1.5 to 40 mm 2 , and even more preferably 2 to 40 mm 2 In a preferred embodiment of the present disclosure, the area is 50 mm 2 Below, 35mm 2 or less, or 15 mm 2 The following is the result.

[0058] From the viewpoint of detection sensitivity or production efficiency, the number of compartments is preferably 1 to 2000, more preferably 4 to 1600, and even more preferably 8 to 400. In a preferred embodiment of the present disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0059] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 The number of cells per 1000 mm is, for example, 50 to 20,000 cells / mm 2 From the viewpoint of cell viability, etc., it is preferably 100 to 15,000 cells / mm 2 , more preferably 100 to 10,000 cells / mm 2 , and more preferably 200 to 10,000 cells / mm 2 , and even more preferably 500 to 7000 cells / mm 2 , particularly preferably 1000 to 5000 cells / mm 2 is.

[0060] The cell chip of the present disclosure generally contains a liquid to prevent the cells from drying out, which is similar to the liquid for suspending the cells described above.

[0061] In one aspect, the present disclosure relates to a method for producing a cell chip, which includes seeding the cells of the present disclosure to form a cell layer on a substrate. In one aspect, the present disclosure relates to a method for preparing cells, which includes storing a cell suspension, and storing the cell suspension at a temperature above the refrigerated storage temperature and below the culture temperature. For these methods, the descriptions of the cells and cell chip of the present disclosure are incorporated by reference.

[0062] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0063] Test Example 1. Preparation of Stably Expressing Cells A transposon vector was introduced into cells derived from the mulberry butterfly (Spilosoma imparilis) (SpIm cells), which contained an olfactory receptor protein (ORA) coding sequence, an olfactory receptor co-receptor coding sequence, a calcium sensor fluorescent protein coding sequence, and a puromycin resistance gene coding sequence under the control of a promoter sequence, with these sequences located between the 5'ITR (inverted repeat) and 3'ITR. Selection was then performed with puromycin to prepare SpIm cells stably expressing an olfactory receptor (hereinafter referred to as ORA cells), in which foreign DNA containing the coding sequence and promoter sequence had been integrated into the chromosomal genomic DNA. The olfactory receptor in question is an insect-derived olfactory receptor and is the receptor for compound a.

[0064] Test Example 2. Cell Preservation Test 1 ORA cells cultured on a cell culture plate were recovered from the plate, and the resulting cell suspension was placed in a 15 ml centrifuge tube and stored under the following conditions. Sf-900III SFM medium was used. A: 4°C (storage for 1 or 7 days). B: 10°C (storage for 1 or 7 days). C: 15°C (storage for 1 or 7 days). D: Storage at 20°C for 1 day, or storage at 20°C for 1 day followed by storage at 4°C for 6 days.

[0065] After storage, the cells were plated in a 384-well plate (384 Greiner TC) at 3.5 × 10 4Cells were seeded at 40 μL per well and placed in a 27°C incubator (without CO2 supply) to allow a cell layer to form on the bottom of the well. 20 hours after seeding, the culture medium was removed from each well, and 40 μL of a solution containing 1% fish gelatin by mass in sf-900 medium was added to each well. Photographs were then taken using a Keyence BZ-X800 microscope.

[0066] The photographs are shown in Figure 1. When cells were stored at refrigeration temperature only (A), there was noticeable cell detachment from the bottom of the well (bright spots), but when storage at temperatures above refrigeration but below the culture temperature was also included (B, C, D), cell detachment was suppressed.

[0067] Test Example 3. Cell preservation test 2 A test was performed in the same manner as Test Example 2, except that the storage conditions were as follows: A: 4°C (storage for 1, 7, or 9 days). B: 10°C (storage for 1, 7, or 9 days). C: Storage at 15°C for 1 day, or storage at 15°C for 1 day followed by storage at 10°C for 6 or 8 days. D: Storage at 20°C for 1 day, or storage at 20°C for 1 day followed by storage at 10°C for 6 or 8 days.

[0068] Photographs are shown in Figure 2. When cells were stored at refrigeration temperature only (A), there was noticeable cell detachment (bright spots) from the bottom of the well, but when storage at temperatures above refrigeration but below the culture temperature was also included (B, C, D), cell detachment was suppressed.

[0069] Test Example 4. Cell preservation test 3 The test was carried out in the same manner as in Test Example 2, except that the storage conditions were as follows: A: Stored at 4°C for 6 days. B: Stored at 10°C for 6 days. C: Stored at 12°C for 6 days. D: Stored at 14°C for 6 days. E: Stored at 16°C for 6 days.

[0070] The photographs are shown in Figure 3. When cells were stored at refrigeration temperature only (A), cell detachment (bright spots) was noticeable, but when storage at temperatures above refrigeration but below the culture temperature was also included (B, C, D), cell detachment was suppressed.

[0071] Test Example 5. Cell Preservation Test 4 Eight SpIm cell lines (#1 to #8) stably expressing olfactory receptors into which different olfactory receptors had been introduced were used, and the test was carried out in the same manner as in Test Example 2, except that the storage conditions were as follows: A: Stored at 4°C for 1 day. B: Stored at 4°C for 7 days. C: Stored at 4°C for 1 day, followed by storage at 10°C for 6 days. D: Stored at 4°C for 8 days. E: Stored at 4°C for 1 day, followed by storage at 10°C for 7 days.

[0072] Photographs are shown in Figure 4. Cell detachment (bright spots) was noticeable when cells were stored at refrigerated temperatures only (B, D). On the other hand, when cells were stored at temperatures above refrigerated temperatures but below the incubation temperature (C, E), cell detachment was suppressed compared to storage at refrigerated temperatures only for the same period (comparisons of A, B, and C, and A, D, and E).

Claims

1. Cells obtained by storing a cell suspension, wherein the storage of the cell suspension comprises storing the cell suspension in a temperature range above the refrigerated storage temperature and below the culture temperature.

2. The cell of claim 1, which is an insect cell.

3. The cells described in claim 2, wherein the temperature range is 6 to 23°C.

4. The cells described in claim 1, wherein the storage time in said temperature range is 12 hours or more.

5. The cells of claim 1, wherein the total storage time of the cell suspension is 12 hours or more.

6. The cells of claim 1, wherein the total storage time of the cell suspension is 400 hours or less.

7. The cells of claim 1, wherein the cell suspension is stored in a non-cell-adherent container.

8. A cell chip comprising the cells according to any one of claims 1 to 7.

9. A method for preparing cells, comprising storing a cell suspension, wherein the cell suspension is stored at a temperature above refrigerated storage temperature and below culture temperature.

10. A method for producing a cell chip, comprising seeding the cells according to any one of claims 1 to 7 to form a cell layer on a substrate.

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