Automated culture system, automated culture method, and extract

Abstract

To provide an automated culture system, an automated culture method, and an extract, which enable high-efficiency medium replacement and efficient recovery of the culture target.SOLUTION: An automated culture system of the present invention includes: an automated culture device and a control device. The automated culture device comprises a culture vessel including a bottomed cylindrical upper chamber equipped with a filter section at its bottom, and a bottomed cylindrical lower chamber disposed on a bottom side of the upper chamber; a first supply unit configured to supply medium into the upper chamber; a first recovery unit that collects medium housed in the upper chamber; and a second recovery unit that collects an extract containing medium housed in the lower chamber and a basement membrane matrix extracted to the lower chamber side from the upper chamber through the filter section. Therein the control device controls a liquid flow in the upper chamber, the lower chamber, the first supply unit, and the first and second recovery units.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an automated culture system, an automated culture method, and an extract. [Background technology]

[0002] The adhesion of epithelial cells to connective tissue is mediated by a basement membrane composed of proteins such as laminin, fibronectin, and type IV collagen. The basement membrane components described above and basement membrane matrices secreted by a mouse sarcoma cell line (EHS cells) have been developed as coating agents for cell culture. For example, a technology for automatically culturing cells by supplying medium is known (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2022-169046 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in the culture method described in Patent Document 1, although the culture medium can be replaced automatically, the target cells are recovered from a mixture with the culture medium, such as by recovering the supernatant, which can result in low efficiency and accuracy of culture medium replacement, and low target recovery efficiency.

[0005] The present invention has been made in view of the above, and aims to provide an automatic culture system, an automatic culture method, and an extract that can perform culture medium replacement and recovery of the culture target with high efficiency. [Means for solving the problem]

[0006] In order to solve the above-mentioned problems and achieve the object, the automatic culture system of the present invention comprises an automatic culture device and a control device that electrically controls the automatic culture device, wherein the automatic culture device comprises a culture vessel having a bottomed cylindrical upper chamber with a filter unit at its bottom, and a bottomed cylindrical lower chamber that is provided on the bottom side of the upper chamber, a first supply unit that supplies culture medium to the upper chamber, a first recovery unit that recovers the culture medium contained in the upper chamber, and a second recovery unit that recovers an extract containing basement membrane substrate that has passed through the filter unit and been extracted from the upper chamber to the lower chamber, and the control device controls the flow of liquid in the upper chamber, the lower chamber, the first supply unit, and the first and second recovery units.

[0007] Furthermore, the automatic culture system according to the present invention is characterized in that, in the above invention, the first supply unit has a first storage unit for storing a culture medium, a first pump, a first tube connecting the first storage unit and the first pump, and a second tube connecting the first pump and the upper chamber; the first recovery unit has a second storage unit for storing the culture medium contained in the upper chamber, a second pump, a third tube connecting the upper chamber and the second pump, and a fourth tube connecting the second storage unit and the second pump; the second recovery unit has a third storage unit for storing the extract, a third pump, a fifth tube connecting the lower chamber and the third pump, and a sixth tube connecting the third storage unit and the third pump; and the control device drives the first to third pumps.

[0008] Moreover, the automatic culture system according to the present invention is characterized in that, in the above invention, it further comprises a second supply unit that supplies a culture medium to the lower chamber.

[0009] In addition, the automatic culture system of the present invention is characterized in that, in the above invention, the second supply unit has a fourth storage unit for storing culture medium, a fourth pump, a seventh tube connecting the fourth storage unit and the fourth pump, and an eighth tube connecting the fourth pump and the lower chamber.

[0010] Furthermore, the automatic culture method according to the present invention is characterized in that in a culture vessel having a bottomed cylindrical upper chamber with a filter section at the bottom and a bottomed cylindrical lower chamber provided on the bottom side of the upper chamber, cells are introduced into the upper chamber containing a culture medium and cultured therein, and an extract containing a basement membrane substrate secreted by the cells during the culture and passed through the filter section and contained in the lower chamber is recovered, the culture medium is supplied to the upper chamber by driving a first pump, the culture medium contained in the upper chamber is recovered by driving a second pump, and the extract is recovered by driving a third pump.

[0011] Moreover, in the automatic culture method according to the present invention, in the above invention, the culture medium is supplied to the lower chamber by driving a fourth pump.

[0012] The extract according to the present invention is characterized in that it is extracted by the automated culture method according to the above invention. [Effects of the Invention]

[0013] The present invention has the effect of enabling the exchange of culture medium and the recovery of the culture subject to be carried out with high efficiency.

[0014] Furthermore, according to the present invention, the use of basement membrane matrices secreted by the junctional epithelial cell line mHAT-JE01, in particular odontogenic ameloblast-associated protein (ODAM), follicular dendritic cell-secreted protein (FDC-SP), and Gm5886 (submandibular androgen-repressed protein (SMARP)) as coating agents has the effect of improving cell adhesion to hydroxyapatite and titanium. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram illustrating cell culture in one embodiment of the present invention. [Figure 2] FIG. 2 is a diagram for explaining teeth and epithelium. [Figure 3] FIG. 3 is a diagram showing a schematic configuration of an automatic culture system according to one embodiment of the present invention. [Figure 4] FIG. 4 shows changes in cell adhesion and cell proliferation in each extract from the upper and lower chambers. [Figure 5] FIG. 5 shows the cell adhesiveness of mHAT-JE01-BM. [Figure 6] FIG. 6 shows the cell adhesiveness of Geltrex. [Figure 7] FIG. 7 shows the cell adhesive properties of gelatin. [Figure 8] FIG. 8 is a diagram illustrating the adhesiveness of epithelial cells to apatite disks. [Figure 9] FIG. 9 is a diagram illustrating the adhesiveness of epithelial cells to titanium discs. [Figure 10] FIG. 10 is a diagram illustrating the adhesiveness of epithelial cells to apatite-coated dishes when the apatite-coated dishes are coated with ODAM and FDC-SP. [Figure 11] FIG. 11 is a diagram illustrating the adhesiveness of epithelial cells to titanium disks when the titanium disks were coated with ODAM and FDC-SP. [Figure 12] FIG. 12 is a diagram illustrating the adhesiveness of epithelial cells to an apatite-coated dish when the apatite-coated dish is coated with Gm5886. [Figure 13] FIG. 13 is a diagram illustrating the adhesiveness of epithelial cells to titanium disks when the titanium disks are coated with Gm5886. [Figure 14] FIG. 14 is a diagram showing a schematic configuration of an automatic culture system according to a modified example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, modes for carrying out the present invention (hereinafter referred to as "embodiments") will be described with reference to the accompanying drawings. Note that the drawings are schematic, and the size relationships between the various parts may differ from the actual ones, and the drawings may include parts whose dimensional relationships and ratios differ from one another.

[0017] (Embodiment) 1 is a diagram illustrating cell culture in one embodiment of the present invention. In this embodiment, a cell line is housed and cultured in a culture vessel 10, and substances secreted from the cell line are collected.

[0018] The culture vessel 10 includes a cylindrical upper chamber 11 with a bottom, and a cylindrical lower chamber 12 with a bottom provided on the bottom side of the upper chamber 11. Although Fig. 1 shows an example in which lids are provided to cover the upper openings of the upper chamber 11 and the lower chamber 12, the vessel may be configured without such lids.

[0019] The upper chamber 11 has a filter section 13 at its bottom. The filter section 13 is constructed using a filter with a plurality of 1-3 μm pores. Therefore, the upper chamber 11 has a bottom with a plurality of pores formed therein, allowing substances with a size smaller than the pore size to pass through. The pore size of the filter section 13 is set to a size that allows the basement membrane matrix secreted by the cell line to be efficiently transferred to the lower chamber 12.

[0020] The lower chamber 12 contains the material that has passed through the filter portion 13 of the upper chamber 11 .

[0021] In this embodiment, the upper chamber 11 contains medium B. 11 The medium B is contained therein. 11 Cell line S 11 (See Fig. 1(a)). 11 The cell line S is adsorbed to the bottom of the upper chamber 11, i.e., the filter part 13, due to polarity or the like. 11 Medium B 11 The cells are cultured under predetermined conditions (temperature, atmosphere) while immersed. 12 will be accommodated.

[0022] Here, the cell line (cell line S 11 The junctional epithelial cell line mHAT-JE01 (see Journal of Oral Biosciences Volume 65, Issue 1, March 2023, pp. 47-54) or the Hertwig's Epithelial Root Sheath (HERS) cell line can be used. Junctional epithelial cells are known to be the cells in the epithelium surrounding the teeth that contact the teeth. HERS cells are known to induce root formation. Both mHAT-JE01 and HERS cell lines secrete a basement membrane matrix, which has the ability to adhere to titanium, apatite, and plastics.

[0023] FIG. 2 is a diagram illustrating teeth and epithelium. Tooth 200 has enamel 202 covering the surface of dentin 201. Enamel 202 is made of hydroxyapatite, a type of calcium phosphate. When fixing an artificial tooth, an implant body (artificial tooth root) is connected to an artificial tooth made of ceramic or the like via an abutment, and the implant body is then bonded to the jawbone using dental materials. In this case, the implant body is made of titanium or the like.

[0024] Furthermore, epithelium 300 exists around the tooth 200. Junctional epithelium 301 of this epithelium 300 comes into contact with (adheres to) the tooth 200. Junctional epithelium 301 is tissue derived from enamel epithelium, and adheres to enamel 202, i.e., hydroxyapatite, via hemidesmosomes. The above-mentioned junctional epithelial cells are the cells that form this junctional epithelium 301. On the other hand, when an artificial tooth is fixed, the artificial tooth or implant body comes into contact with the junctional epithelium 301.

[0025] Cell line S 11 Medium B 11 The basement membrane matrix is ​​secreted by the submerged culture. The secreted basement membrane matrix passes through the filter section 13 and enters the medium B in the lower chamber 12. 12In the case of the mHAT-JE01 cell line, the basement membrane matrix contains components such as odontogenic ameloblast-associated protein (ODAM), follicular dendritic cell-secreted protein (FDC-SP), laminin-332, thrombospondin-1, tenascin, collagen alpha-1 (XVIII) encoding the pro-alpha1 chain of type XVIII collagen, and galectin-1. In the case of the HERS cell line, the basement membrane matrix contains laminin-332, tenascin, collagen alfa-1 (XVIII), periostin, fibronectin, thrombospondin-1, and Fraser extracellular matrix complex subunit 1 (FRAS 1) as components. The inventors have found that among these components, odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) improve epithelial adhesion to apatite and titanium. In addition, the medium B in the lower chamber 12 12 is the medium B that has passed through the filter section 13. 11 or a culture medium that has been previously contained in the lower chamber 12.

[0026] Here, medium B 11 and medium B 12 For example, a medium prepared by mixing DMEM and HamF12 and adding penicillin and streptomycin can be used. 11 In order to enhance the expression of basement membrane substrates, it is preferable to add dexamethasone and / or LPA (lysophosphatidic acid). 11In addition to the above, B-27 (registered trademark), fibroblast growth factor FGF-2, and human epidermal growth factor (EGF) may be added.

[0027] Then, from the lower chamber 12, cell line S 11 Extract S containing basement membrane matrix extracted from 12 The extract S is collected in a tube 100 (see (c) of FIG. 1). 12 By using the basement membrane matrix contained in the above, it is possible to prepare a coating agent that can be applied to dental materials such as hydroxyapatite and titanium to impart cell adhesiveness to teeth and dental materials.

[0028] Next, the automated culture for producing the above-mentioned basement membrane substrate will be described with reference to Fig. 3. Fig. 3 is a diagram showing the schematic configuration of an automated culture system according to one embodiment of the present invention. The automated culture system 1 includes an automated culture device 2 and a control device 3 that electrically controls the automated culture device 2.

[0029] The automatic culture device 2 includes the above-mentioned culture vessel 10, a first supply unit 20, a first recovery unit 30, and a second recovery unit 40.

[0030] The first supply unit 20 has a first storage unit 21, a first pump 22, a first tube 23 connecting the first storage unit 21 and the first pump 22, and a second tube 24 connecting the first pump 22 and the upper chamber 11. The first storage unit 21 stores a medium (medium B) to be supplied to the upper chamber 11. 11 The first supply unit 20 stores the culture medium (culture medium B) stored in the first storage unit 21. 11 ) is sucked through a first tube 23 by driving a first pump 22 and supplied to the upper chamber 11 through a second tube 24.

[0031] The first recovery section 30 has a second storage section 31, a second pump 32, a third tube 33 connecting the upper chamber 11 and the second pump 32, and a fourth tube 34 connecting the second storage section 31 and the second pump 32. The second storage section 31 collects the culture medium (culture medium B) contained in the upper chamber 11. 11 The first collection section 30 contains the culture medium (culture medium B) contained in the upper chamber 11. 11 ) is sucked through the third tube 33 by driving the second pump 32 and sent to the second storage section 31 through the fourth tube 34.

[0032] The second collection unit 40 has a third storage unit 41, a third pump 42, a fifth tube 43 connecting the lower chamber 12 and the third pump 42, and a sixth tube 44 connecting the third storage unit 41 and the third pump 42. The third storage unit 41 stores the basement membrane substrate secreted by the cell line contained in the lower chamber 12. The second collection unit 40 aspirates the basement membrane substrate contained in the lower chamber 12 via the fifth tube 43 by driving the third pump 42, and sends it to the third storage unit 41 via the sixth tube 44. Note that the aspirate may contain, in addition to the basement membrane substrate, some components of the culture medium, etc.

[0033] The control device 3 is a computer configured using one or more hardware components such as a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), an ASIC (Application Specific Integrated Circuit), an FPGA (Field Programmable Gate Array), a CPLD (Complex Programmable Logic Device), etc., and a memory in which various programs, etc. are pre-installed.

[0034] The liquid sending and liquid suction control by each pump is performed by driving each pump under the control of the control device 3.

[0035] The control device 3 drives the pump so that the culture medium flows through the tube at a preset flow rate or flow velocity. The flow rate or flow velocity in the tube that delivers the culture medium and the flow rate or flow velocity in the tube that sucks the culture medium may be the same or different. The flow is adjusted to be suitable for replacing the culture medium. At this time, cell line S 11 is adsorbed to the filter portion 13, so that the medium B 11 During the aspiration, the cell line S is not sucked into the third tube 33 but remains in the upper chamber 11 (filter part 13). 11 In the upper chamber 11, medium B 11 Only the parts can be replaced.

[0036] The control device 3, for example, drives the pump according to a preset schedule to perform medium exchange. In this way, while automatically exchanging the medium in the chamber, the extract containing the basement membrane substrate secreted by the cell line and the used medium are collected. For example, the first supply unit 20 supplies 1000 μL of medium at 10 μL / min twice a day. Collection by the first collection unit 30 may be synchronized with this supply or may be performed according to a separate schedule. Furthermore, collection by the second collection unit 40 may be performed according to the amount of extract, in addition to the preset schedule.

[0037] In addition to the basement membrane matrix secreted by the mHAT-JE01 and HERS cell lines, Gm5886 can also be used as a coating agent. Gm5886 is secreted by the submandibular and lacrimal glands and is known as a submandibular androgen-repressed protein (see "Submandibular androgen-repressed protein (SMARP)"; "A novel mouse protein differentially regulated by androgens in the submandibular and lacrimal glands," Archives of Oral Biology, Volume 52, Issue 6, June 2007, pp. 507-517). However, there have been no reports of its ability to improve epithelial adhesion to apatite or titanium. An extract containing Gm5886 can be obtained, for example, using the automated culture device 2. However, Gm5886 can also be obtained by synthesis or other methods, not limited to automated culture.

[0038] Next, the properties of a coating agent prepared using an extract of the basement membrane matrix secreted by mHAT-JE01 will be described with reference to Figures 4 to 9. The coating agent used was mHAT-JE01-BM, which was prepared using an extract containing the basement membrane matrix obtained by culturing mHAT-JE01.

[0039] Figure 4 shows the changes in cell adhesion and cell proliferation in extracts from the upper and lower chambers. Figure 4 shows the time course of the number of cells adhered to hydroxyapatite in extracts from the upper and lower chambers obtained when cells A and B, extracted from two different cell lines, were cultured. Cells A and B are different mHAT-JE01 individuals. In Figure 4, squares indicate the change in cell number in the upper chamber extract of cell A, circles indicate the change in cell number in the lower chamber extract of cell A, diamonds indicate the change in cell number in the upper chamber extract of cell B, and black squares indicate the change in cell number in the lower chamber extract of cell B. Furthermore, diamonds indicate the change in cell number in the control (commercially available coating agent Geltrex).

[0040] As shown in Figure 4, mHAT-JE01 (cells A and B) have higher cell adhesion than the commercially available coating agent Geltrex, and the rate of cell growth upon culture is also greater. Furthermore, the lower chamber extract has higher cell adhesion than the upper chamber extract, and the rate of cell growth upon culture is also greater.

[0041] Fig. 5 shows the cell adhesiveness of mHAT-JE0101-BM. Fig. 6 shows the cell adhesiveness of Geltrex. Fig. 7 shows the cell adhesiveness of gelatin. Figs. 5 to 7 show the cell adhesiveness of 3 × 10 5 The figure shows the adherent cells when keratinocytes (palatal mucosal epithelium) were seeded at 1000 cells / well onto a 24-well plate and washed with DMEM / HamF12 medium 30 minutes later. As shown in Figures 5 to 7, the cell adhesiveness of mHAT-JE01-BM is higher than that of the commercially available coating agent Geltrex or gelatin, which has the property of adhering to tooth tissue.

[0042] Fig. 8 is a diagram for explaining the adhesiveness of epithelial cells to an apatite disk. Fig. 8 shows the adhesiveness of epithelial cells to an apatite disk. After applying mHAT-JE01-BM as a coating agent to a part of the apatite disk (region R1), 3 × 10 5 This figure shows the adherent cells after keratinocytes (palatal mucosal epithelium) were seeded at 1000 cells / well onto a 24-well plate and washed with DMEM / HamF12 medium 30 minutes later. Note that the keratinocytes are fluorescent (red fluorescent tdTomato), and region R0 was coated with medium only. As shown in Figure 8, region R1, coated with mHAT-JE01-BM, had a high cell count, demonstrating that the coating agent mHAT-JE01-BM exhibits high cell adhesiveness.

[0043] Figure 9 is a diagram illustrating the adhesion of epithelial cells to titanium discs. Figure 9 shows the adherent cells when 1500 / 10 μL of keratinocytes (palatal mucosal epithelium) were seeded on titanium discs coated with mHAT-JE01-BM as a coating agent and on titanium discs without a coating agent, and then washed with DMEM / HamF12 medium 10 minutes later. The cells are fluorescent. As shown in Figure 9, cells were visible on the mHAT-JE01-BM-coated disc even after washing, whereas no cells were visible on the uncoated disc after washing. Application of the coating agent confirmed cell adhesion to the titanium discs.

[0044] Next, the properties of ODAM and FDC-SP as coating agents will be described with reference to FIGS. Fig. 10 is a diagram for explaining the adhesiveness of epithelial cells to apatite-coated dishes when ODAM and FDC-SP are coated on the apatite-coated dishes. Fig. 10 shows the adhesiveness of epithelial cells to apatite-coated dishes when ODAM and FDC-SP are coated on the apatite-coated dishes. 4 The graph shows the change in adherent cells over time when 100 μL of mHAT-PE (a palatal mucosal epithelial cell line) cells were seeded on wells and washed with DMEM / HamF12 60 minutes later. As shown in Figure 10, the cell adhesion of ODAM and FDC-SP was higher than that of uncoated wells, even after the passage of time.

[0045] Fig. 11 is a diagram illustrating the adhesiveness of epithelial cells to titanium disks when the titanium disks are coated with ODAM and FDC-SP. Fig. 11 shows the adhesiveness of epithelial cells at 1 × 10 on titanium disks coated with ODAM and FDC-SP as coating agents, respectively, and on titanium disks without any coating agent. 6The graph shows adherent cells after washing with DMEM / HamF12 medium 60 minutes after seeding mOE-T2 (palatal mucosal epithelium) at 1 / mL. Note that mOE-T2 is fluorescent (red fluorescent tdTomato). As shown in Figure 11, cells were visible on the ODAM- and FDC-SP-coated disks even after washing, whereas no cells were visible on the uncoated disks. Cell adhesion to the titanium disks was confirmed by application of ODAM and FDC-SP.

[0046] Next, the properties of Gm5886 as a coating agent will be described with reference to FIGS. Fig. 12 is a diagram for explaining the adhesiveness of epithelial cells to an apatite-coated dish when the apatite-coated dish is coated with Gm5886. Fig. 12 shows the adhesiveness of epithelial cells to an apatite-coated dish coated with Gm5886 as a coating agent, and to an apatite-coated dish without a coating agent, in which 3.5 × 10 4 The graph shows the change in adherent cells over time when mHAT-PE (a palatal mucosal epithelial cell line) cells / well / 100 μL were seeded and washed with DMEM / HamF12 medium 60 minutes later. As shown in Figure 12, the cell adhesiveness of Gm5886 remains high even after the passage of time, compared to when no coating agent was applied.

[0047] Figure 13 is a diagram illustrating the adhesiveness of epithelial cells to titanium disks when the titanium disks are coated with Gm5886. 6 The graph shows adherent cells after 10 minutes of seeding with mOE-T2 (palatal mucosal epithelium) at 1 / mL and washing with DMEM / HamF12 medium. Note that mOE-T2 is fluorescent (red fluorescent tdTomato). As shown in Figure 13, cells were visible on the Gm5886-coated disk even after washing, while only a few cells were visible on the uncoated disk. Application of Gm5886 confirmed cell adhesion to the titanium disk.

[0048] In the embodiment of the present invention described above, the junctional epithelial cell line mHAT-JE01 is cultured in the automated culture device 2 using a culture vessel 10 having an upper chamber 11 equipped with a filter unit 13 and a lower chamber 12, and the basement membrane matrix secreted by the culture is extracted and recovered through the filter unit 13. During this process, the supply unit and recovery unit automatically replace the culture medium in the upper chamber 11 and the lower chamber 12 under the control of the control device 3. According to this embodiment, the cells and the basement membrane matrix secreted by the cells are reliably separated, and the culture medium is continuously maintained by automatic medium replacement, allowing for highly efficient medium replacement and recovery of the culture target. In contrast, conventional automated culture systems recover the supernatant as described above, resulting in low recovery efficiency.

[0049] Furthermore, when conventional culture methods are used, cells must be passaged periodically, which requires a long period of time (for example, about one month) to obtain the basement membrane matrix secreted by the cell line. However, when the basement membrane matrix is ​​obtained using the automated culture method of the present embodiment, the basement membrane matrix can be obtained in a shorter period of time than the above-mentioned culture methods that require passage.

[0050] Furthermore, according to this embodiment, the basement membrane matrix secreted by mHAT-JE01 during culture, particularly odontogenic ameloblast-associated protein (ODAM), follicular dendritic cell-secreted protein (FDC-SP), and Gm5886, can be used as coating agents to improve cell adhesion to apatite discs (or apatite-coated dishes) and titanium discs.

[0051] (Variation) Next, an automatic culture system according to a modified example of the present invention will be described with reference to Fig. 14. Fig. 14 is a diagram showing the schematic configuration of an automatic culture system according to a modified example of the present invention. An automatic culture system 1A according to the modified example includes an automatic culture device 2A and a control device 3 that electrically controls the automatic culture device 2. Note that the same components as those in the above-described embodiment are assigned the same reference numerals. Below, the parts that are different from the automatic culture system 1 will be described.

[0052] The automatic culture device 2A includes the above-mentioned culture vessel 10, a first supply unit 20, a first recovery unit 30, a second recovery unit 40, and a second supply unit 50.

[0053] The second supply unit 50 has a fourth storage unit 51, a fourth pump 52, a seventh tube 53 connecting the fourth storage unit 51 and the fourth pump 52, and an eighth tube 54 connecting the fourth pump 52 and the lower chamber 12. The fourth storage unit 51 stores a medium (medium B) to be supplied to the lower chamber 12. 13 The second supply unit 50 stores the culture medium (culture medium B) stored in the fourth storage unit 51. 13 ) is sucked through the seventh tube 53 by driving the fourth pump 52, and is supplied to the lower chamber 12 through the eighth tube 54. In addition, medium B 13 Medium B 11 The basement membrane substrate may be the same as or different from the basement membrane substrate.

[0054] In this modification, the control device 3 performs the culture medium exchange by driving the pump according to a preset schedule, for example. 11 In addition to supplying and recovering the basement membrane substrate, a second supply unit 50 supplies a new medium B to the lower chamber 12. 13 In this way, the medium in the chamber is automatically replaced, while the extract containing the basement membrane matrix secreted by the cell line and the spent medium are collected. For example, the second supply unit 50 supplies 1000 μL of medium at a rate of 10 μL / min once every three days. The second collection unit 40 may collect the medium in accordance with this supply, or on a different schedule, or may collect the medium according to the amount of extract.

[0055] In the present modified example described above, similar to the above-described embodiment, the junctional epithelial cell line mHAT-JE01 is cultured in an automatic culture device 2A using a culture vessel 10 having an upper chamber 11 equipped with a filter unit 13 and a lower chamber 12, and the basement membrane matrix secreted by the culture is extracted and recovered through the filter unit 13. Under the control of the control device 3, the supply unit and recovery unit automatically replace the culture medium in the upper chamber 11 and the lower chamber 12. According to this modified example, the cells and the basement membrane matrix secreted by the cells are reliably separated, and the automatic medium replacement allows continuous culture. This allows for highly efficient medium replacement and recovery of the culture target. In contrast, conventional automatic culture systems recover the supernatant as described above, resulting in low recovery efficiency.

[0056] Furthermore, according to this modification, the culture medium B is automatically introduced into the lower chamber 12. 13 Therefore, medium B 13 By adding a component that stabilizes the secreted basement membrane substrate to the solution, the basement membrane substrate can be recovered more stably.

[0057] Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments. For example, the above-described embodiments have been described as examples of recovering basement membrane substrates secreted by junctional epithelial cell lines such as mHAT-JE01 and the HERS cell line. However, the present invention can also be applied to recovering secretions with specific functions. As such, the present invention can include various embodiments not described here. [Explanation of symbols]

[0058] 1. Automated culture system 2, 2A automatic culture device 3. Control device 10 Culture vessel 11 Upper Chamber 12 Lower Chamber 13 Filter section 20 1st supply section 21 First Storage Unit 22 First Pump 23 1st Tube 24 2nd Tube 30 First Collection Section 31 Second Storage Unit 32 Second Pump 33 Third Tube 34 4th Tube 40 Second Collection Section 41 Third Storage Unit 42 Third Pump 43 5th Tube 44 6th Tube 50 2nd supply section 51 4th Storage Unit 52 4th Pump 53 7th Tube 54 8th Tube

Claims

1. An automatic culture device; A control device that electrically controls the automatic culture device; Equipped with The automatic culture device is a culture vessel having a bottomed cylindrical upper chamber, the upper chamber having a filter part provided at the bottom, and a bottomed cylindrical lower chamber provided on the bottom side of the upper chamber; a first supply unit that supplies a culture medium to the upper chamber; a first recovery section that recovers the culture medium contained in the upper chamber; a second collection unit that collects an extract containing basement membrane substrates that have passed through the filter unit and been extracted from the upper chamber to the lower chamber; and the control device controls the flow of liquid in the upper chamber, the lower chamber, the first supply unit, and the first and second recovery units. An automated culture system characterized by:

2. the first supply unit includes a first storage unit that stores a culture medium, a first pump, a first tube that connects the first storage unit and the first pump, and a second tube that connects the first pump and the upper chamber; the first recovery unit includes a second storage unit that stores the culture medium contained in the upper chamber, a second pump, a third tube that connects the upper chamber and the second pump, and a fourth tube that connects the second storage unit and the second pump; the second collection unit includes a third storage unit configured to store the extract, a third pump, a fifth tube connecting the lower chamber and the third pump, and a sixth tube connecting the third storage unit and the third pump; The control device drives the first to third pumps. The automatic culture system according to claim 1 .

3. a second supply unit that supplies a culture medium to the lower chamber; The automatic culture system according to claim 1, further comprising:

4. The second supply unit includes a fourth storage unit that stores a culture medium, a fourth pump, a seventh tube that connects the fourth storage unit and the fourth pump, and an eighth tube that connects the fourth pump and the lower chamber. The automatic culture system according to claim 3 .

5. In a culture vessel having a bottomed cylindrical upper chamber with a filter section at the bottom and a bottomed cylindrical lower chamber provided on the bottom side of the upper chamber, cells are introduced into the upper chamber containing a medium and cultured; a culture for recovering an extract containing the basement membrane matrix secreted by the cells during the culture and contained in the lower chamber through the filter, A culture medium is supplied to the upper chamber by driving a first pump, and the culture medium contained in the upper chamber is collected by driving a second pump; The extract is recovered by driving a third pump. An automatic culture method characterized by:

6. A fourth pump is driven to supply a culture medium to the lower chamber. The automatic culture method according to claim 5 .

7. Extracted by the automatic culture method according to claim 5. An extract characterized by:

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