WO2000017316A1 - Plaque a cupules multiples, pour la congelation de cellules de culture - Google Patents

Plaque a cupules multiples, pour la congelation de cellules de culture Download PDF

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Publication number
WO2000017316A1
WO2000017316A1 PCT/JP1999/005142 JP9905142W WO0017316A1 WO 2000017316 A1 WO2000017316 A1 WO 2000017316A1 JP 9905142 W JP9905142 W JP 9905142W WO 0017316 A1 WO0017316 A1 WO 0017316A1
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WO
WIPO (PCT)
Prior art keywords
cells
plate
cell
freezing
culture
Prior art date
Application number
PCT/JP1999/005142
Other languages
English (en)
Japanese (ja)
Inventor
Kenji Kawamura
Original Assignee
Sumitomo Bakelite Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP26828598A external-priority patent/JP2001252067A/ja
Priority claimed from JP00757699A external-priority patent/JP3587438B2/ja
Application filed by Sumitomo Bakelite Co., Ltd. filed Critical Sumitomo Bakelite Co., Ltd.
Priority to AU57576/99A priority Critical patent/AU5757699A/en
Publication of WO2000017316A1 publication Critical patent/WO2000017316A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates

Definitions

  • the present invention relates to a cell culture multiplate mainly used in the fields of clinical examination, drug discovery, and biotechnology, which is used for a frozen culture cell used in an atsey system using a culture cell, and on a culture substrate.
  • the present invention relates to a multiwell plate for freezing cultured cells that can be used for a cell-based assay system by freezing cells while maintaining the morphological characteristics of the cultured cells, and thawing animal cells that can be stored for a long period of time. . Background art
  • cultured animal cells are stored frozen in liquid nitrogen, and thawed and cultured as needed, but cryopreservation is performed using glass ampules or plastic tubes for freezing.
  • the cells are single cells and are frozen while dispersed in a solution. This is because preserving cells in such a single cell-dispersed state is the most stable method of preserving cells.
  • the cells after thawing the frozen cells, the cells must be adhered to an incubator, and further cultured until the required amount of cells is reached.
  • Japanese Patent Publication No. 5-773989 which omits such labor and time of culture, discloses a method of freezing cultured animal cells in a state of being cultured on a culture substrate.
  • the culture cells were frozen using a 35-cm plastic culture dish, and good results were obtained.
  • this method uses a culture dish and cannot process many samples at once.
  • cells are alive after thawing, but have partially detached from the dish or plate, making it difficult for all cells to remain attached before freezing.
  • the technique of the present invention is not used for culturing cells, but is used in a system in which the cultured cells are used for diagnosis, all living cells must be in the same state. Therefore, measures must be taken to prevent cell leakage.
  • the entire multiwell plate is heated even if it is placed in an incubator at 37 ° C during thawing. Survival rate is extremely poor.
  • the cells at the outer periphery are heated quickly, so that cells The viability is good, but the thawing takes time near the center, resulting in cell viability variations between the multiwell plates and cannot be used to test for cytotoxicity or toxicity.
  • the present invention provides a method of freezing cells cultured on a multiwell plate, increasing the adherence of viable cells for each well in the multiwell plate at the time of thawing, and utilizing the cells as a cell culture or experimental multiwell plate after thawing. It is an object of the present invention to provide a multiwell plate for freezing cultured cells that can be used. Disclosure of the invention
  • the present inventor focused on the heat conductivity of the cell part, which is the cell culture part of the multiwell plate, and thawed efficiently in a short time if the resin thickness of the cell part was within a certain range. However, they found that the survival rate of the cells was dramatically improved, and completed the present invention.
  • the present invention relates to a plastic resin multi-well plate for freezing together with an incubator in a state where cells are cultured, and for thawing at the time of use.
  • the present invention provides a multi-well plate for freezing cultured cells, characterized in that the plate has a 0.2-0.5 repulsion.
  • FIG. 1 is a perspective view showing the positions of a heater and the multi-well plate of the present invention.
  • FIG. 2 is a cross-sectional view of the multiwell plate of the present invention, in which the shell portion and the heating portion contact.
  • FIG. 3 is a perspective view of a heater in which a circulation pipe for heating is set.
  • FIG. 4 is a cross-sectional view of the multi-well plate of the present invention.
  • the reference numerals in the figure are as follows.
  • the multiwell plate of the present invention is a plate having a compartment for culturing a large number of cells, that is, a plate having a large number of cells, which is a cell culture unit.
  • the number of shells in one plate is not particularly limited, but is preferably 48 to 96 mm, and particularly preferably 96 to 100 mm.
  • the cell culture multiplate is formed of a transparent resin such as polystyrene so that observation under a microscope is possible, and generally has a thickness of 1.5 to 2.5 pieces.
  • Simply placing the freeze incubator using this culture vessel in a 37 ° C carbon dioxide incubator takes a long time to thaw, which reduces the viability of the cells after thawing and makes the cells unusable. Become. This tendency is particularly noticeable in the center of the multiwell plate where many wells are concentrated.
  • the present inventor has suggested that the shorter the time required for thawing, the higher the thawing time of the frozen preserved cells to return to room temperature and culture with good viability. It is considered that the same applies to the case in which the thickness of the resin is reduced at the well part of the multi-well plate.
  • the thickness is 0.05 to
  • 0.2 is more preferable in terms of strength and thawing efficiency.
  • the distance between the wells is preferably 2 mm or more, particularly preferably 2 to 3 mm.
  • the plate strength at the outer peripheral portion may be increased. That is, by increasing the thickness of the outer peripheral portion of the molded product plate,
  • the thickness of the resin at the outer peripheral portion is preferably 0.5 to 2.5 sleeps, particularly preferably 1.0 to 2.5 turns.
  • the multiwell plate of the present invention is preferably made of a plastic resin.
  • the plastic resin include a polystyrene resin, a vinyl chloride resin, and a PET resin. Therefore, PET resin is particularly preferred.
  • the heat conductivity is increased, and even if thawing is performed quickly, cells are alive after thawing, but detach from the plate and float in the culture medium, or the cells become sheet-like, Some of the cells may be peeled off and rounded, and in order to prevent such cell peeling, it is necessary to increase the adhesion of the cells to the substrate.
  • There are various methods for improving the adhesiveness between the cells and the substrate in culture but the most reliable and simplest method is to place the cell adhesion factor on the cell adhesion area of the incubator, generally inside the well, especially on the bottom. It is a method of applying or fixing.
  • Cell adhesion factors are proteins and peptides that exist as scaffolds when cells actually exist in the living body. The high affinity and adhesion of the cells to the cells in the living body Proven in stability.
  • the method of applying or immobilizing the cell adhesion factor on the surface of a plastic product consists of simply placing a cell adhesion factor solution of interest in a container and adsorbing the cell adhesion factor on the surface of the container by physical adsorption.
  • There are methods such as introducing a functional group such as a hydroxyl group, a amino group, or a carboxyl group by plasma discharge or treatment with a chemical to immobilize the reactive functional group of the cell adhesion factor.
  • a functional group such as a hydroxyl group, a amino group, or a carboxyl group
  • To apply the cell adhesion factor to the incubator it is possible to dispense a solution containing the cell adhesion factor into a simply molded resin article and leave it for 1 hour to 1 day for physical adsorption.
  • the culture surface of the plate may be subjected to a corona discharge treatment, a plasma treatment, an oxidation treatment, or the like to change the amount of cell adhesion factor and the intensity of the adsorption.
  • a carboxyl group is introduced into the plate surface by carbon monoxide plasma treatment, activated with water-soluble caloformide (WSC), and then immediately treated with an aqueous solution containing a cell adhesion factor such as collagen type I.
  • WSC water-soluble caloformide
  • Cell adhesion factors include collagens such as collagen type I and collagen type IV, fibronectin, gelatin, and cell matrices such as laminin, which forms a basement membrane together with collagen type IV in vivo, and nerve cells.
  • -L-Lysine not only one, but also two or three and a mixture of cell adhesion factors can be used. Since these cell adhesion factors have different adhesion behavior depending on the cells, it is normally best to use a cell adhesion factor suitable for each cell to be cultured. However, as a matter of fact, searching for a suitable cell adhesion factor for individual cells can be a considerable effort by itself. Therefore, a collagen type I coat is preferable except for a combination of a cell and a cell adhesion factor which has already been known (for example, poly-L-uri and nerve cells).
  • the multiwell plate After being thawed, the multiwell plate itself is used in an Atsushi system using cells, and is used directly for measurement of absorbance measurement, chemiluminescence measurement, fluorescence measurement, and colorimetry for detecting enzyme activity. Since it is used directly, a transparent resin or a white or black resin can be used as necessary.
  • the metal block is shaped so that the multi-wall plate can be placed horizontally on top (Fig. 1). At this time, if there is an air layer between the heating element and the multi-well plate, the air layer becomes a buffer, reducing the heat transfer efficiency and increasing the heating time, as shown in Fig. 2.
  • the heating element is processed according to the bottom shape. In the case of a polymer gel, it is important to enclose it in a thin resin container such as a commonly used heat storage material, and to make the multi-well plate a shape that can be placed horizontally. Naturally, contact between the multi-plate and the heating element is a necessary condition. As shown in Fig.
  • the temperature of the heating element may be reduced by passing a pipe through the heating element and circulating hot water or oil at 37 ° C through the pipe. Also, the entire heating unit can be put in a 37 ° C incubator overnight to heat the whole. In order to apply heat to the multi-well plate more effectively, it is necessary to apply heat not only from the bottom surface but also from the side and top surfaces. Instead, it is most effective to have a shape that wraps around the entire multi-wall plate as shown in FIG. Of course, hot water or oil may be circulated through a pipe to suppress the temperature drop.
  • Cultured cells are aseptically cultured in a multi-well plate that has been subjected to steam sterilization, ethylene oxide gas sterilization, radiation sterilization, etc., in an adhered or suspended state.
  • This sterility is an important property that must be maintained during the freezing, storage, and thawing stages. Therefore, the cover of the multiwell plate must not be removed during heating to maintain sterility.
  • a transparent PVC sheet processed into a 96-hole multi-pellet plate (pellet wall thickness 0.1 concealed, outer peripheral wall thickness 0.5 mm, gap between gaps 2 to 3 countries) was UV-sterilized.
  • a 0.3% solution of acidic collagen I type I solution sterilized by filtration with a membrane filter having a pore size of 0.2 ⁇ m, was placed on the bottom of the gel for 1 hour, and collagen was applied.
  • Hep G2 cells a human hepatocellular carcinoma-derived cell line, were transformed into 1 x 10 5 cells ZmL using a culture solution containing 10% fetal bovine serum in Dulbecco's modified Eagle medium (DMEM). After suspending, each plate of the plate was dispensed in 100 ⁇ L portions. (Final cell concentration: 1 ⁇ 10 4 cells / well) After cell seeding, the plate was cultured overnight in a carbon dioxide incubator at 37 ° C. and a carbon dioxide concentration of 5%.
  • DMEM Dulbecco's modified Eagle medium
  • the culture solution is exchanged with a cell cryopreservation medium (a medium containing 10% dimethyl sulfoxide). And the whole cultured cells were frozen in a refrigerator at -20 ° C. After freezing, store in a freezer at 180 ° C. (1) On the third day of storage at 80 ° C in the freezer, remove the frozen plate from the freezer and place it in an incubator at 37 ° C for an aluminum block for heating the plate that has already been heated to 37 ° C. Heated above and thawed frozen. After thawing, the number of cells present in the plate was counted as it was. Further, the inside of the plate is washed three times with 200 ⁇ L of PBS to remove the detached cells and the detached cells from the living cells, and then adhere to the culture surface of the plate. The number of remaining cells was determined.
  • a cell cryopreservation medium a medium containing 10% dimethyl sulfoxide
  • a transparent PVC sheet processed into a 96-hole multi-well plate (wall thickness 0.1 mm, outer peripheral wall thickness 0.5 strokes, gap between gaps 2 to 3 strokes) was UV-sterilized.
  • HepG2 cells a human hepatocellular carcinoma-derived cell line, were cultured at 1 ⁇ 10 5 cells / m in a culture solution of 10% fetal serum added to Dulbecco's modified Eagle medium (DMEM). Then, 100 L of each plate of the plate was dispensed.
  • DMEM Dulbecco's modified Eagle medium
  • the culture solution was replaced with a cell cryopreservation medium, and the cultured cells were frozen in a freezer at -20 ° C. After freezing, store in -80 ° C freezer. -8 (On the 3rd day of storage in the TC freezer, remove the frozen plate from the freezer, heat it at 37 ° C overnight in Incube, and thaw the freeze. The number of cells present in the plate was counted, and the plate was washed three times with 200 ⁇ L of PBS to remove cells that had detached from living cells or cells that had become detached. The number of cells remaining on the surface was determined.
  • HepG2 cells were inoculated in the same manner as in the example, cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, and frozen as in the above example.
  • Example 2 there is no effect on the thawing time in the frozen state, but the cell viability is several percent, but the collagen coated on the surface increases the cell viability. Calculating the viability only by cell adhesion, excluding the cells that have become detached or sparse, the decrease in cell viability is 89 with collagen application and 18% with no collagen application. Was seen. However, from the comparative example level, it is considered that the cell viability in both cases is at a level that is high enough to be used for an assay using cells. Industrial applicability
  • the cells By culturing cells in the multi-well plate for freezing cultured cells of the present invention, the cells can be frozen in a cultured state, and the cells can be frozen and thawed with high viability in a short time when needed, and the number of cells that detach is small. Since experiments using cells can be performed stably, there is no need to wait for the cells to grow or constantly culture cells, which can reduce time and economic waste.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une plaque à cupules multiples, en résine plastique, utilisée pour la congélation de cellules de culture alors qu'elle porte des cellules en incubation et à décongeler avant utilisation. Ladite plaque se caractérise en ce que sa partie cupules pour la mise en culture des cellules est en résine de 0,02 à 0,5 mm d'épaisseur.
PCT/JP1999/005142 1998-09-22 1999-09-21 Plaque a cupules multiples, pour la congelation de cellules de culture WO2000017316A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57576/99A AU5757699A (en) 1998-09-22 1999-09-21 Multiwell plate for freezing cultured cells

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP26828598A JP2001252067A (ja) 1998-09-22 1998-09-22 培養細胞凍結用マルチウェルプレート
JP10/268285 1998-09-22
JP11/7576 1999-01-14
JP00757699A JP3587438B2 (ja) 1999-01-14 1999-01-14 培養細胞凍結用マルチウェルプレート

Publications (1)

Publication Number Publication Date
WO2000017316A1 true WO2000017316A1 (fr) 2000-03-30

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PCT/JP1999/005142 WO2000017316A1 (fr) 1998-09-22 1999-09-21 Plaque a cupules multiples, pour la congelation de cellules de culture

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AU (1) AU5757699A (fr)
WO (1) WO2000017316A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006040871A1 (fr) * 2004-10-12 2006-04-20 Chuo Precision Industrial Co., Ltd. Plaque a puits et dispositif pour cultures cellulaires
KR100718158B1 (ko) * 2006-04-21 2007-05-14 삼성전자주식회사 세포 공동배양 장치
JP2018511315A (ja) * 2015-04-16 2018-04-26 アシンプトート リミテッドＡｓｙｍｐｔｏｔｅ Ｌｔｄ 凍結させる生物学的試料における氷核形成を制御する装置
CN112337525A (zh) * 2020-11-12 2021-02-09 袁明凯 一种用于冻存细胞的操作台
KR20210051979A (ko) * 2019-10-31 2021-05-10 한국해양과학기술원 중형 동물 플랑크톤의 행동학적 반응을 이용한 마비성 패독의 독성 평가 장치 및 그 방법

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06500727A (ja) * 1990-07-18 1994-01-27 マックス　プランク　ガゼルシャフト　ツル　ホルダルング　デル　ヴィッセンシャフテン　エーファウ 化学的及び／又は生化学的及び／又は微生物学的物質を受容する少なくとも１個のくぼみを有するプレート及びこのプレートを製造する方法
JPH06501431A (ja) * 1990-07-18 1994-02-17 マックス　プランク　ガゼルシャフト　ツル　ホルダルング　デル　ヴィッセンシャフテン　エーファウ プレートを製造する方法
JPH06277037A (ja) * 1993-03-24 1994-10-04 Kawasumi Lab Inc 動物組織細胞培養用キット
JPH0751049A (ja) * 1993-08-12 1995-02-28 Fujitsu Ltd 細胞接着試験器具

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06500727A (ja) * 1990-07-18 1994-01-27 マックス　プランク　ガゼルシャフト　ツル　ホルダルング　デル　ヴィッセンシャフテン　エーファウ 化学的及び／又は生化学的及び／又は微生物学的物質を受容する少なくとも１個のくぼみを有するプレート及びこのプレートを製造する方法
JPH06501431A (ja) * 1990-07-18 1994-02-17 マックス　プランク　ガゼルシャフト　ツル　ホルダルング　デル　ヴィッセンシャフテン　エーファウ プレートを製造する方法
JPH06277037A (ja) * 1993-03-24 1994-10-04 Kawasumi Lab Inc 動物組織細胞培養用キット
JPH0751049A (ja) * 1993-08-12 1995-02-28 Fujitsu Ltd 細胞接着試験器具

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006040871A1 (fr) * 2004-10-12 2006-04-20 Chuo Precision Industrial Co., Ltd. Plaque a puits et dispositif pour cultures cellulaires
KR100718158B1 (ko) * 2006-04-21 2007-05-14 삼성전자주식회사 세포 공동배양 장치
JP2018511315A (ja) * 2015-04-16 2018-04-26 アシンプトート リミテッドＡｓｙｍｐｔｏｔｅ Ｌｔｄ 凍結させる生物学的試料における氷核形成を制御する装置
KR20210051979A (ko) * 2019-10-31 2021-05-10 한국해양과학기술원 중형 동물 플랑크톤의 행동학적 반응을 이용한 마비성 패독의 독성 평가 장치 및 그 방법
KR102270265B1 (ko) 2019-10-31 2021-06-25 한국해양과학기술원 중형 동물 플랑크톤의 행동학적 반응을 이용한 마비성 패독의 독성 평가 장치 및 그 방법
CN112337525A (zh) * 2020-11-12 2021-02-09 袁明凯 一种用于冻存细胞的操作台
CN112337525B (zh) * 2020-11-12 2021-08-24 厦门大学附属第一医院 一种用于冻存细胞的操作台

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