JP2019080575A - Somatic cell production system - Google Patents

Abstract

To provide somatic cell production systems capable of producing somatic cells having a certain quality without variation for every worker with low cost and in a short time.SOLUTION: Provided is a somatic cell production system further comprising a pre-introduction cell delivery pathway 20 through which a solution containing pre-introduction cells passes; a factor-introducing device 30 which is connected to the pre-introduction cell delivery channel and introduces a somatic inducing factor into the pre-introduction cells to produce inducing factor-introduced cells; a cell production device 40 which cultures the inducing factor-introduced cells to produce somatic cells; and a housing 200 for storing the pre-introduction cell delivery pathway, factor-introducing device, and cell production device.SELECTED DRAWING: Figure 1

Description

  The present invention relates to somatic cell induction technology, and in particular to a somatic cell production system.

  Embryonic stem cells (ES cells) are stem cells established from human and mouse early embryos. ES cells have pluripotency that can differentiate into all cells present in a living body. Currently, human ES cells are available for cell transplantation therapy for many diseases, such as Parkinson's disease, juvenile diabetes, and leukemia. However, there are also obstacles to transplantation of ES cells. In particular, transplantation of ES cells can elicit an immune rejection similar to the rejection that occurs following unsuccessful organ transplantation. In addition, there are many criticisms and dissent from the ethical point of view on the use of ES cells established by destroying human embryos.

  Under such circumstances, Professor Shingo Yamanaka of Kyoto University has introduced induced pluripotent stem cells (iPS) by introducing four genes: Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells. Succeeded in establishing cells). As a result, Professor Yamanaka received the 2012 Nobel Prize in Physiology and Medicine (for example, see Patent Document 1). iPS cells are ideal pluripotent cells free of rejection and ethical problems. Therefore, iPS cells are expected to be used for cell transplantation therapy. Further, in recent years, a technology has also been established in which another cell can be produced from a specific cell by introducing a specific gene into the cell. Such techniques are expected to be useful for transplantation medicine and drug screening as well as iPS cells.

  Conventionally, there are many methods for converting iPS cells into somatic cells. However, in order to use iPS cells for transplantation therapy, it is important to establish a method for efficiently inducing differentiation of iPS cells. Specifically, the technology used to induce differentiation of iPS cells into somatic cells is established to improve the efficiency and accuracy of differentiation induction, and the functionality of the prepared somatic cells can withstand transplantation treatment There is a need.

  The method for inducing differentiation from iPS cells and embryonic stem cells (ES cells) to somatic cells involves combining hormones and growth factors that determine cell properties, and low molecular weight compounds, and changing their quantitative ratio and concentration over time And, it has been done in a form that mimics the process of development. However, it is difficult to imitate the developmental process completely in vitro and it is inefficient. In addition, as compared to somatic cell differentiation induction in mice, human requires a very long differentiation induction period, for example, three months or more are required to produce a mature nerve. Furthermore, the efficiency of differentiation induction differs greatly depending on the ES / iPS cell line, and there are problems such as the nature of induced somatic cells being uneven.

  Specifically, it has been confirmed that cells induced to differentiate using human ES / iPS cells using a method of utilizing hormones or chemicals are somatic cells in the fetal stage at an early stage. Differentiation induction of human mature somatic cells is extremely difficult and requires long-term culture for several months. However, in drug discovery and transplantation for individuals whose development has been completed, it is very important to produce somatic cells that match the degree of maturity of the individual.

  In addition, although various subtypes of cells exist in nerve cells, methods using hormones or chemicals can not uniformly induce differentiation of these subtype nerves from ES / iPS cells. Therefore, specific neural subtype specific drug discovery screening can not be performed. Therefore, the efficiency of drug discovery screening is low. Also, in transplantation medicine, it is not possible to concentrate and transplant only certain cells with disease.

  On the other hand, a method has been proposed in which a target somatic cell is produced by directly introducing a gene that defines specific somatic cell properties into ES / iPS cells using a virus. Methods using viruses can specifically generate mature neurons in a very short period of time, for example, two weeks, as compared to methods using hormones or chemicals. In addition, when nerve cells are produced by specific gene transfer, for example, only excitatory nerves can be uniformly obtained. Therefore, specific neural subtype specific drug discovery screening is possible, and it is considered that in transplantation medicine, it is possible to concentrate and transplant only specific cells having a disease.

  In addition, when iPS cells are differentiated into somatic cells, undifferentiated iPS cells may remain in the differentiated somatic cells. Therefore, a method of differentiating from one somatic cell to another somatic cell without passing through iPS cells has also been established. Specifically, methods for differentiating fibroblasts into cardiomyocytes and neurons have been developed. These methods are called direct reprogramming and do not go through pluripotent stem cells such as iPS cells, so there is no risk that undifferentiated pluripotent cells remain at the time of transplantation.

Patent No. 4183742

  Somatic cells are established by introducing an inducer such as a gene into cells, expanded, and optionally cryopreserved. However, for example, there are the following problems in producing and industrializing somatic cells (eg, GLP, GMP grade) for clinical use.

1) Cost Somatic cells for clinical use need to be produced and stored in a very clean clean room. However, the cost of maintaining the required level of cleanliness is very high. Therefore, it is costly to produce somatic cells for clinical use, which is a major obstacle to industrialization.

2) Quality The series of tasks from establishment to storage of somatic cells is complicated and often done by hand. In addition, production of somatic cells is owed to individual skills. Therefore, the quality of somatic cells for clinical use may vary depending on the maker or experimental batch.

3) Time In order to prevent cross contamination with cells of others except a specific donor in the clean room, clinical somatic cells derived from only one person are produced in the clean room for a predetermined time. In addition, it takes a long time to establish somatic cells for clinical use and to evaluate their quality. However, since somatic cells for clinical use are produced only for one person at a time in a clean room, it takes a very long time to produce somatic cells for many applicants.

4) Human Resources As mentioned above, at present, preparation of somatic cells for clinical use is largely done by hand. However, few technicians have the necessary skills to be able to produce somatic cells for clinical use.

  On the other hand, an object of the present invention is to provide a somatic cell production system capable of producing somatic cells. The somatic cells are not limited to clinical somatic cells.

  According to the aspect of the present invention, the induction cell is connected to the pre-infusion cell flow channel through which the solution containing the pre-infusion cell solution passes, and the pre-infusion cell flow channel, and the induction cell is introduced There is provided a somatic cell production system comprising a factor introduction device for producing cells, and a cell production device for producing somatic cells by culturing inducer-introduced cells.

  The above-described somatic cell production system may further include a pre-introduction cell fluid delivery channel, a factor delivery device, and a housing for storing the cell production device.

  In the above-described somatic cell production system, somatic cells produced by introducing a somatic inducer may exclude pluripotent stem cells. Somatic cells produced by introducing a somatic inducer may contain differentiated cells. Somatic cells produced by introducing a somatic inducer may contain somatic stem cells. Adult stem cells are also referred to as adult stem cells or tissue stem cells. Somatic cells produced by introducing a somatic inducer may include neural cells. Somatic cells produced by introducing a somatic inducer may include fibroblasts. Somatic cells produced by introducing a somatic inducer may include cardiomyocytes, keratinocytes (keratinocytes), or retinal cells.

  In the above-described somatic cell production system, the pre-introduced cells may contain pluripotent stem cells. Pluripotent stem cells may include ES cells and iPS cells. The pre-introduced cells may contain somatic stem cells. The pre-introduced cells may contain differentiated somatic cells. The pre-introduced cells may contain blood cells. The pre-introduced cells may contain fibroblasts.

  In the above-mentioned somatic cell production system, the cell production apparatus is a somatic cell culture apparatus for culturing the inducer-introduced cells produced by the factor introduction apparatus, and an expansion culture apparatus for expanding the somatic cells established by the somatic cell culture apparatus. And the somatic cell culture apparatus is provided with a first medium replenishing apparatus for replenishing a medium to an induction factor-introduced cell, and the expansion culture apparatus is provided with a second medium supplementing apparatus for replenishing a somatic cell with a culture medium May be

  In the above-described somatic cell production system, the somatic cell culture apparatus may further include a drug supplementing apparatus that supplies a solution containing a drug that kills cells into which the drug resistance factor has not been introduced.

  In the above-mentioned somatic cell production system, the factor introducing device comprises a factor introducing part connected to a cell delivery line before introduction, a factor storage part for storing a somatic inducer, and a cell delivery line before introduction from the factor storage part. Alternatively, the factor introducing part may be provided with a factor delivery line for causing the somatic cell inducing factor to flow, and a pump for causing the liquid in the factor delivery line to flow.

  In the above-described somatic cell production system, the somatic inducer may be DNA, RNA or protein.

  In the factor introduction part of the above-mentioned somatic cell production system, a somatic induction factor may be introduced into the cell before introduction by RNA lipofection.

  In the above-described somatic cell production system, a somatic inducer may be incorporated into the vector. The vector may be a Sendai virus vector.

  The above-described somatic cell production system may further include a packaging device for packaging the somatic cells produced by the cell production device, and the housing may store the packaging device.

  The above-mentioned somatic cell production system is a solution displacing device including a tubular member and a liquid permeation filter disposed inside the tubular member, wherein the tubular member, the cell permeation device on the liquid permeation filter A somatic cell introduction hole for introducing a solution containing somatic cells prepared in step (a), a substitution solution introduction hole for introducing a substitution solution on a liquid permeation filter, and a somatic cell substitution on a liquid permeation filter It may further comprise a solution displacing device provided with a somatic cell outlet for draining the solution and a waste outlet for draining the solution permeating the liquid permeation filter.

  The above-mentioned somatic cell production system further comprises a waste liquid delivery channel connected to the waste liquid discharge port of the solution displacement device, and the flow of the solution in the waste liquid delivery channel is allowed when discarding the solution containing the somatic cell-containing solution. When the somatic cells are dispersed in the replacement solution, the flow of the solution in the waste liquid delivery path may not be allowed.

  In the above-described somatic cell production system, the replacement solution may be a cryopreservation solution.

  The above-described somatic cell production system may further include a separation device for separating pre-introduced cells from blood, and a solution containing the pre-introduced cells separated by the separation device may pass through the pre-introduced cell fluid delivery channel.

  According to the present invention, it is possible to provide a somatic cell production system capable of producing somatic cells.

It is a schematic diagram of the somatic cell manufacturing system which concerns on embodiment of this invention. It is a schematic diagram of the somatic cell manufacturing system which concerns on embodiment of this invention. FIG. 2 is a schematic cross-sectional view of an example of a cell transfer channel of a somatic cell production system according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view of an example of a cell transfer channel of a somatic cell production system according to an embodiment of the present invention. It is a schematic diagram of the culture bag used with the somatic cell manufacturing system which concerns on embodiment of this invention. It is a schematic diagram of the solution displacement device which concerns on embodiment of this invention. It is a schematic diagram of the somatic cell manufacturing system which concerns on embodiment of this invention. 1 is a photograph of cells according to Example 1. 1 is a photograph of cells according to Example 1. 5 is a graph showing the ratio of transfection efficiency and survival rate according to Example 1. FIG. 7 is a photograph of cells according to Example 2. 5 is a graph showing the proportion of TUJ-1 positive cells according to Example 2. FIG. 5 is a graph showing the proportion of TUJ-1 positive cells according to Example 2. FIG. FIG. 7 is a schematic view of a method of transfection according to Example 3. 7 is a photograph of cells according to Example 3.

  Hereinafter, embodiments of the present invention will be described. In the following description of the drawings, the same or similar parts are denoted by the same or similar symbols. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios among the drawings are included.

  The present disclosure includes inventions provisionally filed in the United States (62/356, 199) and for which a foreign application permit has already been issued.

  As shown in FIG. 1, the somatic cell production system according to the embodiment of the present invention is connected to a pre-introduction cell fluid delivery channel 20 and a pre-infusion cell fluid delivery channel 20 through which a solution containing pre-infusion cells passes. A factor introducing device 30 for introducing a somatic inducer into cells before introduction to produce inducer-introduced cells, a cell preparation device 40 for producing somatic cells by culturing inducer-introduced cells, a cell delivery channel before introduction 20, and a case 200 for storing the factor introduction device 30 and the cell preparation device 40.

  The pre-introduced cells are, for example, pluripotent stem cells. As pluripotent stem cells, ES cells and iPS cells can be used. Alternatively, the pre-introduced cells are, for example, differentiated cells. As differentiated cells, somatic stem cells, blood cells, somatic cells differentiated from fibroblasts and the like can be used. Adult stem cells are also referred to as adult stem cells or tissue stem cells.

  Somatic cells produced by introducing a somatic inducer exclude pluripotent stem cells. Somatic cells produced by introducing a somatic inducer are differentiated cells. Examples of differentiated cells include somatic stem cells, nervous system cells, fibroblasts, cardiomyocytes, liver cells, retinal cells, corneal cells, blood cells, keratinocytes (keratinocytes) and chondrocytes. The neural cells may be any of neural cells, neural stem cells and neural progenitor cells. The neural cells may be any of inhibitory neurons, excitatory neurons and dopamine producing neurons. Alternatively, nervous system cells may be motor neurons, oligodendrocyte precursor cells, oligodenrodocytes and the like. Neuronal cells may be MAP2 positive or β-III Tubulin positive.

The somatic cell production system further includes an air purification device that cleans the gas in the housing 200, a temperature management device that manages the temperature of the gas in the housing 200, and carbon dioxide (CO ₂ ) of the gas in the housing 200. ) A carbon dioxide concentration management device may be provided to manage the concentration. The air purification device may include a cleanliness sensor that monitors the cleanliness of the gas in the housing 200. The air cleaner cleans the air in the housing 200 using, for example, a High Efficiency Particulate Air (HEPA) filter, an Ultra Low Penetration Air (ULPA) filter, or the like. The air cleaning device, for example, makes the cleanliness of air in the housing 200 a class of ISO 1 to ISO 6 in ISO standard 14644-1. The thermal management device may include a temperature sensor that monitors the temperature of the gas in the housing 200. CO ₂ concentration control device may be provided with a CO ₂ concentration sensor for monitoring the CO ₂ concentration of the gas within the enclosure 200.

For example, a door or the like is provided in the case 200, but when the door is closed, the inside is completely closed, and the cleanliness, temperature, and CO ₂ concentration of the air inside can be kept constant. It is possible. The housing 200 is preferably transparent so that the state of the internal device can be observed from the outside. In addition, the housing 200 may be a glove box in which gloves such as rubber gloves are integrated.

  The casing 200 may be provided with an introduction port in communication with the cell liquid delivery channel 20 before introduction. A door or the like may be provided in the opening. Alternatively, the inlet may be sealable by a removable sealing material. Cells before introduction are put into the cell delivery channel 20 before introduction from the introduction port. Alternatively, a pre-introduction cell storage container may be disposed in the housing 200 and in communication with the pre-introduction cell liquid flow path 20 for storing the pre-introduction cells.

  Alternatively, as shown in FIG. 2, the somatic cell production system may further include a separation device 10 disposed in the housing 200 for separating pre-introduced cells from blood. In this case, the cell delivery channel 20 before introduction is connected to the separation device 10. A solution containing the pre-introduced cells separated by the separation device 10 passes through the pre-introduced cell delivery channel 20.

  The separation device 10 in the housing 200 receives, for example, a vial containing human blood. The separation device 10 may be, for example, an anticoagulant tank for storing an anticoagulant such as ethylenediaminetetraacetic acid (EDTA), heparin, and biological preparation standard blood storage solution A (ACD-A solution, Terumo Corporation). Have. The separation device 10 adds an anticoagulant to human blood from an anticoagulant tank using a pump or the like.

  In addition, the separation device 10 includes, for example, a separation reagent tank for storing a mononuclear cell separation reagent such as Ficoll-Paque PREMIUM (registered trademark, GE Healthcare Japan Co., Ltd.). The separation apparatus 10 dispenses, for example, 5 mL of the mononuclear cell separation reagent from the separation reagent tank to, for example, two 15 mL tubes using a pump or the like. A resin bag may be used instead of the tube.

  Furthermore, the separation device 10 includes a buffer tank that stores a buffer such as phosphate buffered saline (PBS). The separation apparatus 10 adds and dilutes 5 mL of buffer solutions to 5 mL of human blood, for example, from a buffer solution tank using a pump or the like. Furthermore, the separation apparatus 10 adds diluted human blood in 5 mL each onto the mononuclear cell separation reagent in the tube using a pump or the like.

  The separation device 10 further includes a temperature settable centrifuge. The centrifuge is set to, for example, 18 ° C. The separation device 10 uses a transfer device or the like to place a tube containing a mononuclear cell separation reagent, human blood and the like into the holder of the centrifuge. The centrifuge centrifuges the solution in the tube, for example, at 400 × g for 30 minutes. Instead of the tube, the resin bag may be centrifuged.

  After centrifugation, the separation device 10 collects, with a pump or the like, the white turbid intermediate layer of mononuclear cells of the solution in the tube. The separation device 10 sends out the collected suspension of mononuclear cells to the cell fluid delivery channel 20 before introduction using a pump or the like. Alternatively, further, the separation device 10 adds, for example, 12 mL of PBS to 2 mL of the recovered mononuclear cell solution, and places the tube in the holder of the centrifuge. The centrifuge centrifuges the solution in the tube, for example at 200 × g for 10 minutes.

  After centrifugation, the separator 10 aspirates and removes the supernatant of the solution in the tube using a pump or the like, and 3 mL of mononuclear cell culture medium such as X-VIVO 10 (registered trademark, Ronza Japan Co., Ltd.) is used as a tube. Add to the mononuclear cell solution and suspend. Blood cells may be cultured feeder-free using a basement membrane matrix such as Matrigel (Corning), CELLstart®, ThermoFisher, or Laminin 511 (nippi). The separation device 10 pumps a suspension of mononuclear cells as pre-introduced cells into the pre-introduced cell liquid delivery channel 20 using a pump or the like. The separation device 10 may separate mononuclear cells from blood using a dialysis membrane. In addition, when using pre-introduced cells before introduction, the separation device 10 may be omitted.

  The separation device 10 may separate cells suitable for induction by methods other than centrifugation. For example, if the cells to be separated are T cells, cells positive for any of CD3, CD4 and CD8 may be separated by panning. If the cells to be separated are vascular endothelial precursor cells, cells positive for CD34 may be separated by panning. If the cells to be separated are B cells, cells positive for any of CD10, CD19 and CD20 may be separated by panning. Moreover, you may isolate | separate not only by panning but the magnetic cell separation method (MACS), flow cytometry, etc.

The inner wall of the cell delivery channel 20 before introduction may be coated with poly-HEMA (poly 2-hydroxyethyl methacrylate) to make the cell non-adhesive so that the cells before introduction do not adhere. Alternatively, a material to which the pre-infusion cells do not easily adhere may be used as the material of the pre-infusion cell delivery channel 20. Moreover, for example, by using a material with good temperature conductivity and CO ₂ permeability as the material of the cell delivery line 20 before introduction, the conditions in the cell delivery line 20 before introduction are managed in the housing 200. It is equivalent to the temperature and CO ₂ concentration. Furthermore, from the viewpoint of contamination prevention, the backflow prevention valve may be provided in the cell liquid-sending path 20 before introduction.

  The induction factor delivery mechanism 21 in the housing 200 includes, for example, an induction factor introduction reagent tank for storing an induction factor introduction reagent solution or the like. The induction factor delivery mechanism 21 uses the micropump or the like to suspend the induction factor introduction reagent solution in the induction factor introduction reagent solution, so that the cell delivery path before introduction of the cells in the housing 200 can be suspended. 20 or to the factor introducing device 30.

  An inducer transfer reagent solution such as a gene transfer reagent solution includes, for example, a set of somatic inducer RNA, an RNA transfection solution, and a medium for RNA transfection. RNA transfection involves RNA lipofection. The set of somatic inducer RNAs includes, for example, 100 ng each of the ASCL1 mRNA, Myt1 L mRNA, and neurogenin 2 (Ngn2) mRNA. Ngn2 (neurogenin 2) is a switch protein required for neural cell differentiation.

  The somatic inducer RNA may contain mRNA corresponding to the drug resistance gene. The agents are, for example, antibiotics such as puromycin, neomycin, blasticidin, G418, hygromycin and zeocin. Cells into which mRNA corresponding to a drug resistant gene has been introduced show drug resistance. Somatic inducer RNAs include, for example, Ngn2-T2A-Puro mRNA (Trilink). Cells transfected with Ngn2-T2A-Puro mRNA (Trilink) produce neurogenin2 (Ngn2) and exhibit puromycin resistance.

  The mRNA is capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and may be substituted with 5-methylcytidine and pseudouridine. 5-methyl cytidine and pseudouridine reduce the ability of antibodies to recognize mRNA.

  The RNA transfection solution contains, for example, short interfering RNA (siRNA) or lipofection reagent. As the lipofection reagent for RNA, lipofection reagent for siRNA and lipofection reagent for mRNA can be used. More specifically, Lipofectamine (registered trademark) RNAiMAX (Thermo Fisher Scientific), Lipofectamine (registered trademark) MessengerMAX (Thermo Fisher Scientific), Lipofectamin (registered trademark) 2000, Lipofectamin (registered trademark) 3000 as a lipofection reagent for RNA. Neon Transfection System (Thermo Fisher scientific), Stemfect RNA Transfection Reagent (Stemfect), NextFect (R) RNA Transfection Reagent (Bioo Scientific), Amaxa Registered products) Human T cell Nucleofector (registered product) kit (Lonza, VAPA-1002), Amaxa (registered product) Human CD34 cellNucleofector (registered product) kit (Lonza, VAPA-1003), and ReproRNA (registered trademark) transfection Reagents STEMCELL Technologies), mRNA-in (Thermo fisher scientific) and the like can be used.

  For example, the factor transfer device 30 may suspend the cells in the culture solution after introducing the somatic inducer into the cells. The factor transfer device 30 may perform transfection of somatic inducers multiple times. For example, after a predetermined time such as 24 hours after introducing the somatic inducer into the cells, the medium may be changed, and the cells may be transfected again with the somatic inducer. Furthermore, transfection of somatic inducers into cells and cell culture for a predetermined time may be repeated multiple times, for example, two to four times.

When lipofection of somatic cell induction factor RNA, for example, when using a 12-well plate, the number of cells per well is 1 × 10 ⁴ to 1 × 10 ⁸ , 5 × 10 ⁴ to 1 × It is 10 ⁶ or 1 × 10 ⁵ to 5 × 10 ⁵ . The bottom area of one well is 4 cm ² . The amount of somatic inducer RNA during lipofection of somatic inducer RNA is 200 to 5000 ng, 400 to 2000 ng, or 500 to 1000 ng per time. The amount of lipofection reagent during lipofection of somatic inducer RNA is 0.1 μL to 100 μL, 1 μL to 50 μL, or 1.5 μL to 10 μL.

  The medium used for lipofection of somatic inducer RNA is, for example, a low serum medium such as Opti-MEM (registered trademark, Gibco). The culture medium used during and after lipofection of somatic inducer RNA may contain B18R protein. B18R protein attenuates the cell's innate antiviral response. B18R proteins may be used to suppress cell death associated with the immune response associated with the insertion of RNA into cells. However, when the cells are differentiated into somatic cells in a short period of time, the medium may not contain B18R protein, or may contain B18R protein at a low concentration such as 0.01% to 1%.

  Animal cells differentiate into somatic cells within 10 days, 9 days, 8 days, or 7 days of lipofection of somatic inducer RNA. When the somatic cells to be produced are nervous system cells, whether or not they are differentiated into nervous system cells is confirmed by whether or not β-IIITubulin, MAP2, or PsA-NCAM is positive. (beta) -IIITubulin, MAP2, PsA-NCAM, and vGlu are markers which label nerve cells, and are constituent proteins of microtubules in nerve cell processes.

  Alternatively, an inducer transfer reagent solution such as a gene transfer reagent solution may contain, for example, a Sendai virus vector solution. Although Sendai virus-derived RNA is not integrated into the host DNA, the host can introduce a gene of interest. The Sendai virus vector set includes, for example, the mRNA of ASCL1, the mRNA of Myt1L, and the mRNA of Ngn2 such that the multiplicity of infection (MOI) is 0.01 to 1000, 0.1 to 1, or 1 to 10. The inducer Sendai virus vector may contain mRNA corresponding to the drug resistance gene. The inducer RNA contained in the Sendai virus vector includes, for example, Ngn2-T2A-Puro mRNA (Trilink). The introduction of Sendai virus into cells may be performed once.

  The factor introducing device 30 uses a pump or the like to send out a solution containing cells into which an inducing factor has been introduced (inducing factor introduced cells) to the introduced cell delivery channel 31.

The inner wall of the introduced cell delivery channel 31 in the housing 200 may be coated with poly-HEMA so as not to adhere to the inducer-introduced cells so as not to adhere. Alternatively, a material to which the induction factor-introduced cells are less likely to adhere may be used as the material of the introduced cell delivery channel 31. Also, for example, by using a material with good temperature conductivity and CO ₂ permeability as the material of the introduced cell liquid flow path 31, the conditions in the introduced cell liquid flow path 31 are controlled in the housing 200. It becomes equivalent to temperature and CO ₂ concentration. Furthermore, from the viewpoint of contamination prevention, the backflow prevention valve may be provided in the introduced cell delivery channel 31. Further, as shown in FIG. 3, one or more weirs may be provided in the introduced cell liquid flow path 31 so as to intermittently change the inner diameter. Alternatively, as shown in FIG. 4, the inner diameter of the introduced cell delivery channel 31 may be changed intermittently.

  As shown in FIG. 1 and FIG. 2, the cell preparation device 40 connected to the introduced cell delivery channel 31 includes a somatic cell culture device 50 for culturing the inducer-introduced cells prepared by the factor introduction device 30, and a somatic cell. A first dividing mechanism 60 for dividing a cell mass (cell colony) consisting of somatic cells established by the culture device 50 into a plurality of cell masses, an expansion culture device 70 for expanding culture of somatic cells, and a growth culture device 70 The system includes a second dividing mechanism 80 for dividing a cell mass consisting of expanded somatic cells into a plurality of cell masses, and a somatic cell transfer mechanism 90 for sequentially sending somatic cells to the package device 100. However, for example, when no cell mass is formed or when it is not necessary to divide the cell mass, the first division mechanism 60 and the second division mechanism 80 may be omitted.

  The somatic cell culture apparatus 50 may be equipped with culture vessels such as well plates, bags, and tubes inside. The somatic cell culture apparatus 50 may also be equipped with a pipetting machine. The somatic cell culture apparatus 50 receives the solution containing the somatic cell induction factor-introduced cells from the introduced cell delivery channel 31, and distributes the solution to the culture vessel with a pipetting machine.

  When differentiating the cells into neural cells, the somatic cell culture apparatus 50 puts N2 medium (DMEM / F12, 25 μg) which is a neural differentiation medium, for example, on days 1 to 7 after placing the induction factor-introduced cells in the culture vessel. / ML insulin, 50 μg / mL human transferrin, 30 nmol / L sodium selenite, 20 nmol / L progesterone, 100 nmol / L putrescine) is added to the culture vessel. The medium may be supplemented with ROCK inhibitor (Selleck) at a concentration of 10 μmol / L for several days.

  After distributing the inducer-introduced cells into the culture vessel, the somatic cell culture apparatus 50 performs medium exchange, for example, on the ninth day, and performs medium exchange thereafter until target cells such as neural cells are observed. Note that changing the medium also includes partially replacing the medium and supplying the medium.

  In the somatic cell culture apparatus 50, drug selection may be performed to kill the cells into which the drug resistance factor has not been introduced. When the somatic inducer RNA contains mRNA corresponding to the drug resistance gene, the drug-resistant inducer-transduced cells selectively survive by supplying a solution containing the drug to the culture vessel. For example, if the somatic inducer RNA contains an mRNA corresponding to a puromycin resistance gene, exposing the cells after lipofection to puromycin kills the cells other than those into which the somatic inducer RNA has been introduced. It is possible to select cells into which somatic inducer RNA has been introduced. The drug may be contained in the medium. The concentration of the drug is, for example, 2 mg / L.

  In the somatic cell culture apparatus 50, inducer-transduced cells are cultured using a medium containing a drug that kills cells to which the drug resistance factor has not been introduced for a predetermined period, and then induction is performed using a drug-free medium The factor-transduced cells may be cultured.

When the target somatic cell is formed, the somatic cell culture apparatus 50 recovers the somatic cell with a pipetting machine. Furthermore, the somatic cell culture apparatus 50 places the container containing the recovered somatic cells in an incubator, and causes the somatic cells to react with the trypsin alternative recombinant enzyme at 37 ° C., 5% CO ₂ for 10 minutes. When the cell mass is physically broken, the trypsin alternative recombinant enzyme may not be present. For example, the somatic cell culture apparatus 50 breaks the somatic cell mass by pipetting with a pipetting machine. Alternatively, the somatic cell culture apparatus 50 passes cells through a cell mass through a pipe provided with a filter or a pipe whose internal diameter is intermittently changed, like the introduced cell delivery channel 31 shown in FIG. 3 or FIG. 4. The mass may be broken.

  For example, in the case of inducing a nerve, the somatic cell culture apparatus 50 then adds the neural differentiation medium as described above to the solution containing the somatic cell mass crushed.

The culture in the somatic cell culture apparatus 50 may be performed not in a well plate but in a bag. The bag may be CO ₂ permeable. The culture may be adhesion culture or suspension culture. In the case of suspension culture, agitation culture may be performed. Furthermore, the culture in the somatic cell culture apparatus 50 may be a hanging drop culture.

  The somatic cell culture apparatus 50 may be provided with a first medium replenishing apparatus for replenishing a culture vessel such as a well plate, a bag and a tube with a culture medium containing a culture solution. The first medium replenishing device may recover the culture solution in the culture vessel, filter the culture solution using a filter or a dialysis membrane, and reuse the purified culture solution. At that time, a growth factor or the like may be added to the culture solution to be recycled. In addition, the somatic cell culture device 50 may be provided with a drug supply device for supplying a culture container with a solution containing a drug that kills cells into which the drug resistance factor has not been introduced. Furthermore, the somatic cell culture apparatus 50 may further include a temperature management apparatus that manages the temperature of the culture medium, a pH management apparatus that manages the pH of the culture medium, a humidity management apparatus that manages the humidity near the culture medium, and the like.

In the somatic cell culture apparatus 50, for example, cells are placed in a culture solution permeable bag 301 such as a dialysis membrane as shown in FIG. 5, and the culture solution permeable bag 301 is made into a culture solution impermeable bag 302. The culture solution may be put into the bags 301, 302. Bag 302 may be a CO ₂ permeability, it may not be CO ₂ permeability. The somatic cell culture apparatus 50 prepares a plurality of bags 302 containing fresh culture fluid, and for each predetermined period, the outer bag 302 containing the bag 301 containing cells contains fresh culture fluid. The bag may be replaced with a bag 302.

  A first somatic cell fluid delivery channel 51 is connected to the somatic cell culture apparatus 50 shown in FIGS. 1 and 2. The somatic cell culture apparatus 50 sends out a solution containing somatic cells to the first somatic cell fluid delivery channel 51 using a pump or the like. In addition, the first somatic cell fluid delivery channel 51 has an inner diameter that allows only induced cells smaller than a predetermined size to pass, and is connected to a branch channel that removes uninduced cells of a predetermined size or more. It may be

The inner wall of the first somatic cell delivery channel 51 may be coated with poly-HEMA so as to be non-cell-adhesive so that somatic cells do not adhere. Alternatively, as the material of the first somatic cell fluid delivery channel 51, a material in which somatic cells do not easily adhere may be used. In addition, by using a material having a good temperature conductivity and a CO ₂ permeability as the material of the first somatic cell fluid delivery channel 51, the conditions in the first somatic cell fluid transport channel 51 can be controlled in the housing 200. It is equivalent to the temperature and CO ₂ concentration. Furthermore, a backflow prevention valve may be provided in the first somatic cell fluid delivery channel 51 from the viewpoint of contamination prevention.

  The first somatic cell fluid delivery channel 51 is connected to the first dividing mechanism 60. The first dividing mechanism 60 includes, for example, a mesh. The cell mass contained in the solution is divided into a plurality of cell masses of the size of each pore of the mesh when passing through the mesh by water pressure. For example, if the size of each pore of the mesh is uniform, the sizes of the plurality of cell masses after division will be substantially uniform. Alternatively, the first dividing mechanism 60 may include a nozzle. For example, by finely processing the inside of the substantially conical nozzle in a step-like manner, the cell mass contained in the solution is divided into a plurality of cell masses when passing through the nozzle.

  An expansion culture apparatus 70 is connected to the first dividing mechanism 60. The solution containing the somatic cell mass divided by the first division mechanism 60 is sent to the expansion culture apparatus 70. When the cell mass is not formed, the first dividing mechanism 60 may be omitted. In this case, the first somatic cell delivery channel 51 is connected to the expansion culture apparatus 70.

The expansion culture apparatus 70 can store, for example, a well plate inside. Moreover, the expansion culture apparatus 70 is equipped with a pipetting machine. The expansion culture apparatus 70 receives the solution containing the somatic cells from the first dividing mechanism 60 or the first somatic cell delivery channel 51, and distributes the solution to the wells with a pipetting machine. After distributing the somatic cells in the wells, the expansion culture apparatus 70 cultures somatic cells at, for example, about 8 days at 37 ° C. and 5% CO ₂ . Moreover, the expansion culture apparatus 70 performs culture medium exchange suitably.

When a cell mass is formed, the expansion culture apparatus 70 adds a trypsin alternative recombinant enzyme such as TrypLE Select (registered trademark, Life Technologies) to the cell mass. Furthermore, the expansion culture apparatus 70 raises the temperature of the container containing the cell mass, and makes the cell mass react with the trypsin replacement recombinant enzyme at 37 ° C., 5% CO ₂ for 1 minute. When the cell mass is physically broken, the trypsin alternative recombinant enzyme may not be present. For example, the expansion culture apparatus 70 breaks cell clumps by pipetting with a pipetting machine. Alternatively, the expansion culture apparatus 70 may be provided with a cell mass through a cell mass through a pipe provided with a filter or a pipe whose internal diameter is intermittently changed as in the introduced cell delivery channel 31 shown in FIG. 3 or 4. You may break it. Thereafter, the expansion culture apparatus 70 shown in FIGS. 1 and 2 adds a medium such as a maintenance culture medium to the solution containing the cell mass. Furthermore, in the case of the adhesion culture, the expansion culture apparatus 70 peels the cell mass from the container with an automatic cell scraper or the like, and the solution containing the cell mass is transferred to the first dividing mechanism 60 via the expansion culture liquid flow channel 71. Send to

The culture in the expansion culture apparatus 70 may be performed in a bag or a tube instead of the well plate. The bag or tube may be permeable to CO ₂ . The culture may be adhesion culture, suspension culture or hanging drop culture. In the case of suspension culture, agitation culture may be performed.

  The expansion culture apparatus 70 may be provided with a second medium replenishing apparatus for replenishing the culture solution to a culture vessel such as a well plate, a bag and a tube. The second medium replenishing device may recover the culture solution in the culture vessel, filter the culture solution using a filter or a dialysis membrane, and reuse the purified culture solution. At that time, a growth factor or the like may be added to the culture solution to be recycled. Further, the expansion culture apparatus 70 may further include a temperature control apparatus that controls the temperature of the culture medium, a humidity management apparatus that controls the humidity near the culture medium, and the like.

Also in the expansion culture apparatus 70, for example, cells are placed in a culture solution permeable bag 301 such as a dialysis membrane as shown in FIG. 5, and the culture solution permeable bag 301 is made into a culture solution impermeable bag 302. The culture solution may be put into the bags 301, 302. The bag 302 may be CO ₂ permeable. The expansion culture apparatus 70 prepares a plurality of bags 302 containing fresh culture fluid, and the outer bag 302 containing the bag 301 containing cells contains fresh culture fluid at predetermined intervals. The bag 302 may be replaced.

  The somatic cell production system shown in FIGS. 1 and 2 may further include an imaging device for imaging the culture in the somatic cell culture device 50 and the expansion culture device 70. Here, when a colorless medium is used as the medium used in the somatic cell culture apparatus 50 and the expansion culture apparatus 70, it is possible to suppress diffuse reflection and autofluorescence which may occur when a colored medium is used. However, in order to confirm the pH of the culture medium, a pH indicator such as phenol red may be included. In addition, since the shape and size of the cells are different between the induced cells and the non-induced cells, the somatic cell production system captures the cells in the somatic cell culture apparatus 50 and the expansion culture apparatus 70. The apparatus may further comprise an induction state monitoring device that calculates the proportion of induced cells. Alternatively, the induction status monitoring device may specify the proportion of cells induced by antibody immunostaining or RNA extraction. Furthermore, the somatic cell production system may be equipped with an uninduced cell removal device that removes uninduced cells by magnetic cell separation method, flow cytometry, or the like.

  The cell mass divided by the first dividing mechanism 60 shown in FIGS. 1 and 2 is again cultured in the expansion culture apparatus 70. The division of cell mass in the first dividing mechanism 60 and the culture of somatic cells in the expansion culture apparatus 70 are repeated until the required amount of cells is obtained. In addition, as above-mentioned, when not forming a cell mass, 1st division | segmentation mechanism 60 may be abbreviate | omitted.

  A second somatic cell fluid delivery channel 72 is connected to the expansion culture apparatus 70. The expansion culture apparatus 70 sends out the solution containing the expanded culture somatic cells to the second somatic cell liquid delivery channel 72 using a pump or the like. However, in the case of suspension culture, exfoliation is unnecessary. The second somatic cell fluid delivery channel 72 has an inner diameter that allows only induced somatic cells smaller than a predetermined size to pass, and is connected to a branch channel that removes uninduced cells having a predetermined size or more. May be

The inner wall of the second somatic cell delivery channel 72 may be coated with poly-HEMA so as to be non-cell-adhesive so that somatic cells do not adhere. Alternatively, the material of the second somatic cell delivery channel 72 may be a material that is hard to adhere to somatic cells. Further, by using a material having a good temperature conductivity and a CO ₂ permeability as the material of the second somatic cell fluid delivery channel 72, the conditions in the second somatic cell fluid delivery channel 72 can be managed in the housing 200. It is equivalent to the temperature and CO ₂ concentration. Furthermore, a backflow prevention valve may be provided in the second somatic cell fluid delivery channel 72 from the viewpoint of contamination prevention.

  The second somatic cell fluid delivery channel 72 is connected to the second dividing mechanism 80. The second dividing mechanism 80 includes, for example, a mesh. The cell mass contained in the solution is divided into a plurality of cell masses of the size of each pore of the mesh when passing through the mesh by water pressure. For example, if the size of each pore of the mesh is uniform, the sizes of the plurality of cell masses after division will be substantially uniform. Alternatively, the second dividing mechanism 80 may include a nozzle. For example, by finely processing the inside of the substantially conical nozzle in a step-like manner, the cell mass contained in the solution is divided into a plurality of cell masses when passing through the nozzle.

  The second cell dividing mechanism 80 shown in FIG. 2 is connected to a somatic cell transfer mechanism 90 for sequentially sending somatic cells to the package device 100. When the cell mass is not formed, the second dividing mechanism 80 may be omitted. In this case, the second somatic cell delivery channel 72 is connected to the somatic cell transport mechanism 90.

  A pre-package cell flow channel 91 is connected between the somatic cell transfer mechanism 90 in the housing 200 and the packaging device 100. The somatic cell transfer mechanism 90 sequentially sends somatic cells to the packaging device 100 via the pre-package cell flow path 91 using a pump or the like.

The pre-package cell channel 91 may be coated with poly-HEMA so that somatic cells do not adhere. Alternatively, the material of the pre-package cell flow channel 91 may be a material to which body cells do not easily adhere. In addition, by using a material with good temperature conductivity and CO ₂ permeability as the material of the pre-package cell flow channel 91, the condition in the pre-package cell flow channel 91 is the controlled temperature in the housing 200 and It becomes equivalent to CO ₂ concentration. The pre-package cell flow channel 91 may be provided with a backflow prevention valve from the viewpoint of contamination prevention.

  The pre-package cell flow channel 91 is connected to a cryopreservation liquid delivery mechanism 110. The cryopreservation solution delivery mechanism 110 delivers the cell cryopreservation solution to the pre-package cell channel 91. Thus, the somatic cells are suspended in the cell cryopreservation solution in the pre-package cell flow channel 91.

  The packaging apparatus 100 sequentially freezes the somatic cells sent via the pre-package cell flow channel 91. For example, the packaging apparatus 100 places somatic cells in a cryopreservation container such as a cryotube every time the somatic cells are received, and instantaneously freezes the solution containing the somatic cells at, for example, -80 ° C or lower. If a cryopreservation container with a small surface area per volume tends to take a long time to freeze, it is preferable to use a cryopreservation container with a large surface area per volume. By using a cryopreservation container having a large surface area per volume, it is possible to increase cell viability after thawing. The shape of the cryopreservation container includes, but is not limited to, capillary and spherical shapes. Also, depending on the required cell viability after thawing, it is not always necessary to do the snap freezing.

  For freezing, for example, a vitrification method is used. In this case, DAP 213 (Cosmo Bio Inc.) and Freezing Medium (Reprocel Co., Ltd.) can be used as a cell cryopreservation solution. Freezing may be carried out by a conventional method other than the vitrification method. In this case, as a cell cryopreservation solution, CryoDefend-Stem Cell (R & D system company), STEM-CELL BANKER (registered trademark, Nippon Zenyaku Kogyo Co., Ltd.), etc. can be used. The freezing may be performed by liquid nitrogen or by a Peltier device. Use of a Peltier element makes it possible to control temperature change and to suppress temperature unevenness. The packaging apparatus 100 carries the cryopreservation container out of the housing 200. If the frozen cells are for clinical use, the cryopreservation container is preferably a completely closed system. However, the packaging apparatus 100 may be packaged in a storage container without freezing somatic cells.

  Alternatively, in the packaging apparatus 100, the solvent of the solution containing the somatic cells may be replaced with the cryopreservation solution from the medium using the solution replacement device 101 as shown in FIG. Inside the solution displacing device 101, a filter 102 is provided on the bottom surface with pores through which somatic cells do not permeate. In addition, the somatic cell introduction hole to which the first liquid transfer channel 103 for transferring the medium containing the somatic cells is connected to the solution displacing device 101 on the filter 102 inside, the somatic cells on the filter 102 inside. A substitution solution inlet hole connected to a second liquid feed flow path 104 for feeding a frozen liquid not containing the liquid, and a first discharge flow path 105 for discharging a frozen liquid containing somatic cells from above the filter 102 inside Somatic cell outflow holes are provided. Furthermore, the solution displacing device 101 is provided with a waste liquid discharge port to which a second discharge flow path 106 for discharging the solution that has permeated the filter 102 is connected. A tube or the like can be used for each of the first liquid transfer flow path 103, the second liquid transfer flow path 104, the first discharge flow path 105, and the second discharge flow path 106.

  First, as shown in FIGS. 6A and 6B, with the flow of the solution in the second discharge channel 106 stopped, the inside of the solution displacing device 101 from the first liquid delivery channel 103 Put in the medium containing somatic cells. Next, as shown in FIG. 6C, the flow of the solution in the second discharge channel 106 is allowed, and the culture medium is discharged from the solution displacement device 101. At this time, as shown in FIG. 6 (d), somatic cells remain on the filter 102. Furthermore, as shown in FIGS. 6E and 6F, with the flow of the solution in the second discharge channel 106 stopped, the inside of the solution displacing device 101 from the second liquid delivery channel 104 Add the cryopreservation solution, and disperse somatic cells in the cryopreservation solution. Thereafter, as shown in FIG. 6 (g), the cryopreservation solution containing somatic cells is drained from the first drainage channel 105. The cryopreservation solution containing somatic cells is sent to a cryopreservation container or the like via the first discharge channel 105.

  The somatic cell production system shown in FIGS. 1 and 2 may further include a sterilizer for sterilizing the inside of the housing 200. The sterilizer may be a dry heat sterilizer. In this case, the wiring of devices using electricity, such as the separation device 10, the cell fluid delivery channel 20 before introduction, the induction factor fluid delivery mechanism 21, the factor delivery device 30, the cell preparation device 40, and the package device 100, is heat resistant. It is preferable that it is the wiring which it has. Alternatively, the sterilizer may sterilize the inside of the housing 200 by releasing a sterilizing gas such as ozone gas, hydrogen peroxide gas, or formalin gas into the housing 200.

  The somatic cell production system was recorded by the operation of the separation device 10, the cell delivery channel 20 before introduction, the induction factor delivery mechanism 21, the factor delivery device 30, the cell preparation device 40, the package device 100, etc., and the imaging device. The image may be transmitted to an external server by wire or wirelessly. Furthermore, the external server controls the separation device 10 of the somatic cell production system, the induction factor delivery mechanism 21, the factor introduction device 30, the cell production device 40, the packaging device 100, etc. based on the standard operation procedure (SOP). It may be monitored whether each device is operating based on SOP, and operation records of each device may be generated automatically.

  According to the somatic cell production system described above, somatic cells can be induced automatically.

  The somatic cell production system according to the embodiment is not limited to the configuration shown in FIGS. 1 and 2. For example, in the somatic cell production system according to the embodiment shown in FIG. 7, blood is fed from the blood storage unit 201 to the mononuclear cell separation unit 203 via the blood delivery channel 202. As the blood storage unit 201 and the mononuclear cell separation unit 203, for example, a tube can be used. The blood delivery channel 202 is, for example, a resin tube, a silicon tube, or the like. The same applies to the other liquid feed paths described later. The blood storage unit 201 may be provided with an identifier such as a bar code to manage blood information. The pump 204 is used for liquid feeding. As the pump 204, a positive displacement pump can be used. Examples of positive displacement pumps include reciprocating pumps, including piston pumps, plunger pumps, and diaphragm pumps, or rotary pumps, including gear pumps, vane pumps, and screw pumps. Examples of diaphragm pumps include tubing pumps and piezoelectric (piezo) pumps. Examples of tubing pumps include Perister Pump (registered trademark, Atto Co., Ltd.), and RP-Q1 and RP-TX (Takasago Electric Co., Ltd.). Examples of piezoelectric pumps include SDMP 304, SDP 306, SDM 320, and APP-20KG (Takasago Electric Co., Ltd.). In addition, a microfluidic chip module (Takasago Electric Co., Ltd.) in which various types of pumps are combined may be used. By using a closed pump such as Perister Pump (registered trademark), a tubing pump, and a diaphragm pump, it is possible to pump blood without direct contact with the blood inside the blood delivery channel 202. The same applies to the other pumps described later. Alternatively, a syringe pump may be used as the pump 204 and the pump 207, the pump 216, the pump 222, the pump 225, the pump 234, the pump 242, and the pump 252 described later. Even pumps other than sealed pumps can be reused by heat sterilization and the like.

  An erythrocyte coagulating agent is sent to the mononuclear cell separation unit 203 from the separation agent storage unit 205 via the liquid feeding path 206 and the pump 207. For example, a tube can be used as the separating agent storage unit 205. An identifier such as a barcode may be attached to the separating agent storage unit 205 to manage information of the separating agent. As the erythrocyte coagulating agent, for example, HetaSep (registered trademark, STEMCELL Technologies) or an erythrocyte coagulating agent (Nipro) can be used. In the mononuclear cell separation unit 203, red blood cells are sedimented by the red blood cell coagulant, and mononuclear cells are separated. The supernatant containing the mononuclear cells in the mononuclear cell separation unit 203 is sent to the mononuclear cell purification filter 210 via the mononuclear cell liquid flow path 208 and the pump 209. In the mononuclear cell purification filter 210, components other than mononuclear cells are removed, and a solution containing mononuclear cells as cells before introduction is obtained. As the mononuclear cell purification filter 210, Purecell (registered trademark, PALL), Cell soba E (Asahi Kasei Corporation), Sepacel PL (Asahi Kasei Corporation), Adacolumn (registered trademark, JIMRO), and separation bag (Nipro Corporation) Etc. can be used.

  In FIG. 7, the mononuclear cell separation unit 203, the separating agent storage unit 205, the mononuclear cell purification filter 210, the pumps 204, 207, 209, and the like constitute a separation device. However, as described above, when using pre-introduced cells before introduction, the separation device may be omitted.

  The solution containing the pre-introduced cells is sent to the factor introducing unit 213 via the pre-introduced cell fluid delivery channel 211 and the pump 212. For example, a tube can be used as the factor introducing unit 213. The somatic inducer is sent to the factor introducing unit 213 from the factor storage unit 214 including the somatic inducer via the factor feed path 215 and the pump 216. For example, a tube can be used as the factor storage unit 214. The factor storage unit 214 may be provided with an identifier such as a barcode to manage information of the somatic factor. The factor storage unit 214, the pump 216, and the like constitute an induction factor delivery mechanism. In the factor introducing unit 213 as a factor introducing device, a somatic inducer is introduced into a cell by, for example, an RNA lipofection method, and an inducer-transduced cell is produced. However, the method of transfection of the inducer is not limited to the RNA lipofection method. For example, Sendai virus vectors containing somatic inducers may be used. Alternatively, the somatic factor may be a protein. Alternatively, induction factor transfection may be performed multiple times over several days.

The inducer-introduced cells are sent to the somatic incubator 219 as part of the cell preparation apparatus via the introduced cell delivery channel 217 and the pump 218. The introduced cell delivery channel 217 is, for example, temperature permeable and CO ₂ permeable. In the somatic cell culture vessel 219, the drug is supplied from the cell culture medium storage unit 220 containing the drug-containing cell culture medium for the first several days after the introduction of the somatic inducer into the cells, via the medium delivery channel 221 and the pump 222. Containing cell culture medium is supplemented. The drug-containing cell culture medium contains a drug that kills cells to which no drug resistance factor has been introduced. The medium delivery channel 221 is, for example, temperature permeable and CO ₂ permeable. An identifier such as a barcode may be attached to the drug-containing cell culture medium storage unit 220 to manage information of the drug-containing cell culture medium. The drug-containing cell culture medium storage unit 220, the culture medium delivery channel 221, and the pump 222 constitute a culture medium replenishment device.

Thereafter, the somatic cell culture device 219 is replenished with the somatic cell culture medium from the somatic cell culture medium storage unit 223 containing the somatic cell culture medium suitable for the target somatic cell, via the culture medium delivery channel 224 and the pump 225. . An identifier such as a barcode may be attached to the somatic cell storage unit 223 to manage information of the somatic medium. The medium delivery channel 224 is, for example, temperature permeable and CO ₂ permeable. The somatic cell storage unit 223, the medium delivery channel 224, and the pump 225 constitute a medium supply device.

  The drug-containing cell culture medium storage unit 220 and the somatic cell culture medium storage unit 223 may be stored cold at a low temperature such as 4 ° C., for example, in the cold storage unit 259. The culture medium sent from the drug-containing cell culture medium storage unit 220 and the somatic cell culture medium storage unit 223 may be sent to the incubator after being heated to 37 ° C. by a heater outside the cold storage unit 259, for example. Alternatively, the temperature of the surrounding medium of the liquid supply channel may be set so that the temperature of the culture medium which has been cryopreserved is raised to 37 ° C. while advancing the liquid supply channel. The old culture medium in the somatic cell culture device 219 is sent to the waste fluid storage section 228 via the waste fluid delivery channel 226 and the pump 227. An identifier such as a bar code may be attached to the waste liquid storage unit 228 to manage information on the waste liquid.

The somatic cells cultured in the somatic cell culture vessel 219 are transferred via the introduced cell delivery channel 229, the pump 230, and optionally the cell mass divider 231 to a first expansion incubator 232 as part of a cell preparation apparatus. Sent to By passing through the cell mass divider 231, the cell mass is divided into smaller cell masses. If no cell mass is formed, the cell mass divider 231 may be omitted. The first expansion incubator 232 is replenished with the somatic medium from the somatic medium storage unit 223 including the somatic medium via the medium delivery channel 233 and the pump 234. The introduced cell delivery channel 229 and the medium delivery channel 233 are, for example, temperature permeable and CO ₂ permeable. The somatic cell storage unit 223, the medium delivery path 233, and the pump 234 constitute a medium supply device.

  The aged culture medium in the first expansion incubator 232 is sent to the waste fluid storage section 228 via the waste fluid delivery channel 235 and the pump 236.

The somatic cells cultured in the first expansion incubator 232 pass through a cell transfer channel 237, a pump 238, and optionally a cell mass divider 239, and then a second expansion incubator as part of a cell preparation apparatus. It is sent to 240. By passing through cell mass divider 239, the cell mass is divided into smaller cell masses. If no cell mass is formed, the cell mass divider 239 may be omitted. The second expansion incubator 240 is replenished with the somatic cell culture medium from the somatic cell culture medium storage unit 223 including the somatic cell culture medium via the culture medium delivery channel 241 and the pump 242. The introduced cell delivery channel 237 and the medium delivery channel 241 are, for example, temperature permeable and CO ₂ permeable. The somatic cell storage unit 223, the culture medium delivery channel 241, and the pump 242 constitute a culture medium supply device.

  The aged culture medium in the second expansion incubator 240 is sent to the waste fluid storage section 228 via the waste fluid delivery channel 243 and the pump 244.

  The somatic cells cultured in the second expansion incubator 240 are sent to the solution substitution device 247 through the introduced cell delivery channel 245 and the pump 246. The solution displacer 247 may have, for example, the configuration shown in FIG. In the solution replacer 247 shown in FIG. 7, the somatic cells are held by the filter, and the culture medium is sent to the waste fluid storage section 228 via the waste fluid delivery channel 248 and the pump 249.

  After stopping the flow of the solution in the waste liquid feed path 248 by stopping the operation of the pump 249 or after closing the waste liquid feed path 248 with a valve or the like, the solution displacing unit 247 contains a frozen stock solution containing a frozen stock solution. From the storage unit 250, a cryopreservation solution is introduced via the liquid delivery path 251 and the pump 252. This disperses somatic cells in the cryopreservation solution.

  The cryopreservation solution in which the somatic cells are dispersed is sent into the cryopreservation container 255 via the liquid feed path 253 and the pump 254 as part of the package device. The cryopreservation container 255 is stored in the low temperature storage 256. For example, liquid nitrogen at −80 ° C. is sent from the liquid nitrogen storage 257 to the low temperature storage 256 via the liquid feed path 258. Thereby, the somatic cells in the cryopreservation container 255 are frozen. However, freezing of somatic cells may not depend on liquid nitrogen. For example, the low temperature storage 256 may be a freezer such as a compression freezer, an absorption freezer, or a Peltier freezer. In addition, if freezing is unnecessary, the somatic cells need not be frozen.

A backflow prevention valve may be provided as appropriate in the above-described liquid feed path. The above-described liquid delivery path, mononuclear cell separation unit 203, mononuclear cell purification filter 210, factor introduction unit 213, somatic cell incubator 219, first expansion incubator 232, second expansion incubator 240, and solution replacement The container 247 and the like are stored in, for example, a cassette-like case 260 made of resin or the like. The case 260 is made of, for example, a sterilizable heat resistant material. The inside of the case 260 is in an environment suitable for cell culture, for example, 37 ° C., 5% CO ₂ concentration. The liquid flow path through which the culture medium flows is made of, for example, a CO ₂ permeable material. However, the case 260 is not limited to the cassette shape. For example, it may be a flexible bag. In addition, the above-described liquid flow path, mononuclear cell separation unit 203, mononuclear cell purification filter 210, factor introduction unit 213, somatic cell incubator 219, first expansion incubator 232, second expansion incubator 240, and The solution displacing device 247 or the like may be divided into a plurality of cases and stored.

  Case 260 is disposed in housing 200. Pump, blood storage unit 201, separating agent storage unit 205, factor storage unit 214, drug-containing cell culture medium storage unit 220, somatic cell culture medium storage unit 223, waste liquid storage unit 228, cryopreservation container 255, low temperature storage 256, and liquid The nitrogen storage 257 is disposed inside the housing 200 and outside the case 260.

  The case 260 and the housing 200 have, for example, fitting portions that fit each other. Therefore, the case 260 is disposed at a predetermined position in the housing 200. In the casing 200, a pump, a blood storage unit 201, a separating agent storage unit 205, a factor storage unit 214, a drug-containing cell culture medium storage unit 220, a somatic cell culture storage unit 223, a waste solution storage unit 228, a cryopreservation container 255 The low temperature storage 256 and the liquid nitrogen storage 257 are placed at predetermined positions. When the case 260 is disposed at a predetermined position in the housing 200, the liquid delivery path in the case 260 includes the pump, the blood storage unit 201, the separating agent storage unit 205, the factor storage unit 214, and the drug-containing cell culture medium storage unit. 220, somatic cell culture medium storage unit 223, waste solution storage unit 228, cryopreservation container 255, low temperature storage 256, and liquid nitrogen storage 257.

  For example, the case 260 and its inclusions are disposable and may be discarded and replaced with new ones once somatic cell freezing is complete. Alternatively, when the case 260 and its inclusion are reused, an identifier such as a bar code may be attached to the case 260 to manage the number of times of use and the like.

  According to the somatic cell production system according to the embodiment described above, it is possible to automatically produce somatic cells from the cells before introduction without human intervention.

(Other embodiments)
As described above, although the present invention has been described by the embodiment, it should not be understood that the description and the drawings, which form a part of this disclosure, limit the present invention. Various alternative embodiments, embodiments and operation techniques should be apparent to those skilled in the art from this disclosure. For example, the factor transfer device 30 may induce cells by transfection using a virus vector such as retrovirus, lentivirus and Sendai virus, a plasmid, or protein transfection. Alternatively, the factor transfer device 30 may induce cells by electroporation. In addition, the pre-infusion cell delivery channel 20, the introductory cell delivery channel 31, the first somatic cell delivery channel 51, the expansion culture delivery channel 71, the second somatic cell delivery channel 72, and the pre-package cell flow channel 91 It may be provided on the substrate by microfluidics technology. As such, it should be understood that the present invention includes various embodiments and the like not described herein.

Example 1
Prepare 12-well dishes coated with a solubilized basement membrane preparation (Matrigel, Corning) and use ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor (Selleck) at a concentration of 10 μmol / L in each well. ) In a feeder-free medium (mTeSR.RTM. 1, STEMCELL Technologies). ROCK inhibitors suppress cell death.

The iPS cells were dispersed in tissue / cultured cell detachment / separation / dispersion solution (Accutase, Innovative Cell Technologies) and seeded in 12-well dishes. Cells to be transfected were plated at a density of 4 × 10 ⁵ cells per well. The bottom area of one well was 4 cm ² . Untransfected control cells were plated at a density of 2 × 10 ⁵ cells per well. The cells were then cultured in feeder free medium for 24 hours. At this time, the temperature was 37 ° C., the CO ₂ concentration was 5%, and the oxygen concentration was 25% or less.

  Mix 1.25 ml of Xeno-Free Medium (Pluriton, STEMGENT), 0.5 μl of Pluriton Supplement (STEMGENT), and 2 μl of B18R recombinant protein-containing solution (eBioscience) at a concentration of 100 ng / μl, and use transfection medium as Prepared. Prior to transfection, the feeder-free medium in each well was replaced with transfection medium, and the cells were cultured at 37 ° C. for 2 hours.

  Green fluorescent protein (GFP) mRNA (TriLink) was prepared. The mRNA was capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and substituted with 5-methylcytidine and pseudouridine.

  Further, 1.5 mL of microcentrifuge tubes A and 1.5 mL of microcentrifuge tubes B were prepared for each well.

  Add 62.5 μL of low serum medium (Opti-MEM®, Gibco) to tube A, add 1.875 μL of mRNA transfer reagent (Lipofectamine MessengerMax®, Invitrogen) to it, mix well This was the first reaction solution. Tube A was then tapped so that the first reaction mixture was mixed for 10 minutes at room temperature.

  62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco) was added to tube B, 500 ng of GFP mRNA (Trilink) was added, and mixed well to form a second reaction solution.

  The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped to form liposomes for 5 minutes at room temperature. Next, the mixed reaction solution was added to each well and allowed to stand overnight at 37 ° C. This added 500 ng of GFP mRNA to each well.

  When the cells were observed the next day using a fluorescence microscope, as shown in FIGS. 8 and 9, the coloration of the transfected cells was confirmed. Furthermore, as shown in FIG. 10, cell viability was confirmed. From this, it was shown that it is possible to introduce mRNA into iPS cells using lipofection reagent and RNA and to express protein.

(Example 2)
Prepare 12-well dishes coated with a solubilized basement membrane preparation (Matrigel, Corning) and use ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor (Selleck) at a concentration of 10 μmol / L in each well. ) In a feeder-free medium (mTeSR.RTM. 1, STEMCELL Technologies). ROCK inhibitors suppress cell death.

The iPS cells were dispersed in tissue / cultured cell detachment / separation / dispersion solution (Accutase, Innovative Cell Technologies) and seeded in 12-well dishes. Cells to be transfected were plated at a density of 4 × 10 ⁵ cells per well. Untransfected control cells were plated at a density of 2 × 10 ⁵ cells per well. The cells were then cultured in feeder free medium for 24 hours.

  Mix 1.25 ml of Xeno-Free Medium (Pluriton, STEMGENT), 0.5 μl of Pluriton Supplement (STEMGENT), and 2 μl of B18R recombinant protein-containing solution (eBioscience) at a concentration of 100 ng / μl, and use transfection medium as Prepared. Prior to transfection, the feeder-free medium in each well was replaced with transfection medium, and the cells were cultured at 37 ° C. for 2 hours.

  Ngn2-T2A-Puro mRNA (Trilink) and green fluorescent protein (GFP) mRNA (Trilink) were prepared. The mRNA was capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and substituted with 5-methylcytidine and pseudouridine. In addition, mRNA is purified on a silica membrane, and is carried out by using a solution containing 1 mmol / L of sodium citrate at pH 6 as a solvent together with a reagent for mRNA introduction (Lipofectamine MessengerMax (registered trademark), Invitrogen). Further, 1.5 mL of microcentrifuge tubes A and 1.5 mL of microcentrifuge tubes B were prepared in the same number as the number of wells.

  Add 62.5 μL of low serum medium (Opti-MEM®, Gibco) to tube A, add 1.875 μL of mRNA transfer reagent (Lipofectamine MessengerMax®, Invitrogen) to it, mix well This was the first reaction solution. Tube A was then tapped so that the first reaction mixture was mixed for 10 minutes at room temperature.

  Put 62.5 μL of low serum medium (Opti-MEM®, Gibco) into tube B, add 500 ng of Ngn2-T2A-Puro mRNA (Trilink) and 1500 ng of GFP mRNA (Trilink) to it. It mixed and it was set as the 2nd reaction liquid.

  The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped to form liposomes for 5 minutes at room temperature. Next, the mixed reaction solution was added to each well and allowed to stand overnight at 37 ° C. Thus, 500 ng of Ngn2 mRNA and 100 ng of GFP mRNA were added to each well.

  When observed on the first day after mRNA introduction, as shown in FIG. 11, cell coloration was confirmed.

  Nerve Differentiation Medium (N2 / DMEM /), containing medium for each of the following 2 days, every day, ROCK inhibitor (Selleck) at a concentration of 10 μmol / L and antibiotic (puromycin) at a concentration of 1 mg / L. The cells were completely exchanged with F12 / NEAA (Invitrogen), and cells transfected with mRNA were selected. On the third day, the medium was replaced with neural differentiation medium (N2 / DMEM / F12 / NEAA, Invitrogen) containing B18R recombinant protein-containing solution (eBioscience) at a concentration of 200 ng / mL. Thereafter, by the seventh day, the medium was replaced by half with the same medium.

  On the seventh day, the medium was removed from the wells and washed with 1 mL PBS. Thereafter, 4% PFA was added and reacted at 4 ° C. for 15 minutes for fixation. After washing twice with PBS, the primary antibody was diluted with 5% CCS, 0.1% Triton in PBS medium, and 500 μL was added. As primary antibodies, rabbit anti-human Tuj1 antibody (BioLegend 845501) and mouse anti-rat and human Ngn2 antibodies (R and D Systems) are used, and rabbit anti-human Tuj1 antibody (BioLegend 845501) is diluted 1/1000 in buffer, or Mouse anti-rat and human Ngn2 antibodies (R and D Systems) were diluted 1/75 in buffer, and 1 / 10,000 dilution of DAPI in buffer was added to each well and allowed to react at room temperature for 1 hour. Tuj1 antibody is an antibody against β-III Tubulin.

  After one hour of reaction at room temperature, 1 mL of PBS was added to each well, and the wells were well mixed, and then the PBS was discarded. Add PBS again, discard and use 1/1000 dilution of donkey anti-mouse IgG (H + L) secondary antibody Alexa Fluor® 555 complex (Thermofisher) at 1/1000 dilution of donkey anti-rabbit in permeation buffer 500 μL of a secondary antibody-containing permeation buffer containing an IgG (H + L) secondary antibody Alexa Fluor (registered trademark) 647 complex (Thermofisher) was added and allowed to react at room temperature for 30 minutes.

  After 30 minutes of reaction at room temperature, the cells were washed twice with PBS, observed with a fluorescence microscope, and the cells emitting fluorescence were counted.

  FIG. 12 shows that Ngn2-T2A-Puro mRNA is introduced using lipofection, and after 2 days of culture after adding puromycin, the cells are cultured for 5 days without addition of puromycin and stained with Tuji1 for fluorescence microscopy It is the photograph observed in. FIG. 13 shows the percentage of TUJ-1 positive cells on day 7 after transfection of Ngn2-T2A-Puro mRNA using each transfection reagent according to the above procedure. From these results, it was shown that nerve cells were induced.

(Example 3)
Prepare 12-well dishes coated with a solubilized basement membrane preparation (Matrigel, Corning) and use ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor (Selleck) at a concentration of 10 μmol / L in each well. ) In a feeder-free medium (mTeSR.RTM. 1, STEMCELL Technologies).

The iPS cells were dispersed in tissue / cultured cell detachment / separation / dispersion solution (Accutase, Innovative Cell Technologies) and seeded in 12-well dishes. Cells to be transfected were plated at a density of 4 × 10 ⁵ cells per well. Untransfected control cells were plated at a density of 1 × 10 ⁵ cells per well. The cells were then cultured in feeder free medium for 24 hours. At this time, the temperature was 37 ° C., the CO ₂ concentration was 5%, and the oxygen concentration was 25% or less.

  B18R-containing transfection by mixing 1.25 mL of Xeno-free medium (Pluriton, STEMGENT), 0.5 μL of Pluriton Supplement (STEMGENT), and 2 μL of B18R recombinant protein-containing solution (eBioscience) at a concentration of 100 ng / μL The medium was prepared. Also, B18R-free transfection medium was prepared by mixing 1.25 mL of Xeno-free medium (Pluriton, STEMGENT) and 0.5 μL of Pluriton Supplement (STEMGENT).

  Before transfection, the feeder free medium in each well was replaced with B18R-containing transfection medium or B18R-free transfection medium, and the cells were cultured at 37 ° C. for 2 hours.

  Ngn2-T2A-Puro mRNA (Trilink) and GFP mRNA (Trilink) were prepared. The mRNA was capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and substituted with 5-methylcytidine and pseudouridine.

  Further, 1.5 mL of microcentrifuge tubes A and 1.5 mL of microcentrifuge tubes B were prepared in the same number as the number of wells.

  Add 62.5 μL of low serum medium (Opti-MEM®, Gibco) to tube A, add 1.875 μL of mRNA transfer reagent (Lipofectamine MessengerMax®, Invitrogen) to it, mix well This was the first reaction solution. Tube A was then tapped so that the first reaction mixture was mixed for 10 minutes at room temperature.

  Add 62.5 μL of low serum medium (Opti-MEM®, Gibco) to tube B, add 500 ng of Ngn2-T2A-Puro mRNA (Trilink) and 100 ng of GFP mRNA (Trilink) to it. It mixed and it was set as the 2nd reaction liquid.

  The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped to form liposomes for 5 minutes at room temperature. Next, the mixed reaction solution was added to each well and allowed to stand overnight at 37 ° C. Thus, 500 ng of Ngn2 mRNA and 100 ng of GFP mRNA were added to each well. Moreover, as shown in FIG. 14, the thing which performed transfection once, twice, and 3 times was prepared.

  Nerve Differentiation Medium (N2 / DMEM /), containing medium for each of the following 2 days, every day, ROCK inhibitor (Selleck) at a concentration of 10 μmol / L and antibiotic (puromycin) at a concentration of 1 mg / L. The cells were completely exchanged with F12 / NEAA (Invitrogen), and cells transfected with mRNA were selected. On the third day, the medium was replaced with neural differentiation medium (N2 / DMEM / F12 / NEAA, Invitrogen) containing B18R recombinant protein-containing solution (eBioscience) at a concentration of 200 ng / mL. Thereafter, by the seventh day, the medium was replaced by half with the same medium.

  On the seventh day, the medium was removed from the wells and washed with 1 mL PBS. Thereafter, 4% PFA was added and reacted at 4 ° C. for 15 minutes for fixation. After washing twice with PBS, 50 μL of a primary antibody diluted with a permeation buffer containing 5% CCS and 0.1% Triton X in PBS was added to each well and allowed to react at room temperature for 1 hour. The primary antibody was a 1: 1000 dilution of mouse anti-human Tuj1 antibody (BioLegend 84501) and a 1: 150 dilution of mouse anti-human Ngn2 antibody (R and D Systems, MAB3314-SP) to 1: 150. Yes, and DAPI was added to be 1: 10,000.

  One hour later, 1 mL of PBS was added to each well and the wells were well mixed, and then the PBS was discarded. Add PBS again, discard and use donkey anti-mouse IgG (H + L) secondary antibody Alexa Fluor® 555 complex (Thermofisher, A-21428) 1: 1000 in donkey anti-rabbit IgG H + L) 500 μL of a secondary antibody-containing permeation buffer containing 1: 1000 secondary antibody Alexa Fluor (registered trademark) 647 complex (Thermofisher, A31573) was added, and reacted at room temperature for 30 minutes.

  The cells were washed twice with PBS, observed with a fluorescence microscope, and the cells emitting fluorescence were counted. As a result, as shown in FIG. 15, GFP was hardly expressed on day 9 when mRNA was transfected only once. On the other hand, those transfected with mRNA 3 times expressed GFP even on day 9. From this, it was revealed that mRNA was degraded in cells, and protein expression was transient.

  As described above, it was shown that after seeding iPS cells, RNA can be transfected and induced into neurons in several days. In addition, since it can be induced to nerve cells in a short period of time, it was shown that the medium does not need to contain the B18R protein that is usually used to suppress the cell death associated with the immune reaction accompanying the insertion of RNA into cells.

  2 ... tube, 10 ... separation device, 20 ... cell delivery path before introduction, 21 ... induction factor delivery mechanism, 30 ... factor introduction device, 31 ... introduction cell delivery Path 40: cell preparation device 50: somatic cell culture device 51: somatic cell liquid delivery channel 60: division mechanism 70: expansion culture device 71: expansion culture Fluid delivery channel 72: somatic cell fluid delivery channel 80: division mechanism 90: somatic cell transport mechanism 91: pre-package cell flow channel 100: packaging device 101 · Solution displacing unit · 102 · · · · · · · · · · · · · · · · · · · · solution substitution device, 102 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 103 Storage fluid delivery mechanism, 200 ... housing, 201 ... blood storage part, 202 ... blood delivery path, 203 ... single Sphere separation unit, 204: pump, 205: separation agent storage unit, 206: liquid feed path, 207: pump, 208: mononuclear cell liquid feed path, 209: pump, 210 ··· mononuclear cell purification filter, 211 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Factor introduction part, 214 · · · · · · · · · · · · · Path: 216: pump, 217: introduced cell delivery path, 218: pump, 219: somatic cell incubator, 220: cell culture medium storage section, 221: culture medium delivery path , 222: pump, 223: somatic cell culture medium storage unit, 224: medium delivery channel, 225: pump, 226: waste liquid delivery channel, 227: pump, 228 .. · Waste liquid storage section, 229 ... introduced cell liquid delivery path, 230 ... pump, 231 · · · Cell mass divider 232 ... Enlarge incubator, 233 ... medium liquid feeding passage, 234 ... pump, 235 ... waste liquid feeding passage, 236 ... pump, 237 ... transduced cells feed Liquid path, 238: pump, 239: cell mass divider, 240: enlarged incubator, 241: medium feed path, 242: pump, 243, waste liquid feed path, 244 ... pump, 245 ... introduction cells liquid feeding passage, 246 ... pump, 247 ... solution replacer, 248 ... waste liquid feeding passage, 249 ... pump, 250 ... freeze Storage liquid storage unit, 251 ... liquid transfer path, 252 ... pump, 253 ... liquid transfer path, 254 ... pump, 255 ... frozen storage container, 256 ... low temperature storage, 257 ... Liquid nitrogen storage, 258 ... Liquid transfer path, 259 ... Refrigerated storage unit, 260 ... case, 301 ... bag, 302 ... bag

Claims (21)

A pre-infusion cell delivery channel through which a solution containing pre-infusion cells passes;
A factor introducing device connected to the pre-introduction cell flow channel and introducing a somatic inducer into the pre-introduction cell to produce an inducer-introduced cell;
A cell preparation apparatus for culturing somatic cells by culturing the inducer-introduced cells;
A somatic cell production system comprising:   The somatic cell production system according to claim 1, further comprising a housing for storing the pre-introduction cell liquid flow path, the factor introduction device, and the cell preparation device.   The somatic cell production system according to claim 1, wherein the somatic cells exclude pluripotent stem cells.   The somatic cell production system according to claim 1 or 2, wherein the somatic cell comprises a differentiated cell.   The somatic cell production system according to claim 1, wherein the somatic cells include somatic stem cells.   The somatic cell production system according to claim 1, wherein the somatic cells include nervous system cells.   The somatic cell production system according to claim 1, wherein the pre-introduction cell comprises a pluripotent stem cell.   The somatic cell production system according to claim 1 or 2, wherein the pre-introduction cell comprises a somatic stem cell.   The somatic cell production system according to claim 1, wherein the pre-introduction cell comprises a differentiated somatic cell. The cell preparation device is
A somatic cell culture apparatus for culturing the inducer-introduced cells produced by the factor-introducing apparatus;
An expansion culture apparatus for expanding and culturing somatic cells established by the somatic cell culture apparatus;
Equipped with
The somatic cell culture apparatus comprises a first medium supplementing apparatus for replenishing a medium to the inducer-introduced cells;
The expansion culture apparatus comprises a second medium supplementing apparatus that supplements the somatic cells with a medium.
The somatic cell production system according to any one of claims 1 to 9.   11. The somatic cell production system according to claim 10, wherein the somatic cell culture apparatus further comprises a drug supplementation apparatus that supplies a solution containing a drug that kills cells into which the drug resistance factor has not been introduced. The factor introducing device is
A factor introducing unit connected to the cell delivery channel before introduction;
A factor storage unit for storing the somatic cell induction factor,
A factor delivery line for flowing the somatic cell induction factor from the factor storage unit to the pre-introduction cell flow channel or the factor introduction section;
A pump for flowing the liquid in the factor delivery channel;
The somatic cell production system according to any one of claims 1 to 11, comprising:   The somatic cell production system according to claim 12, wherein the somatic cell induction factor is DNA, RNA or protein.   The somatic cell production system according to claim 12, wherein the somatic cell induction factor is introduced into the pre-introduced cell by RNA lipofection in the factor introduction part.   The somatic cell production system according to claim 12, wherein the somatic cell induction factor is incorporated into a vector.   The somatic cell production system according to claim 15, wherein the vector is a Sendai virus vector.   The somatic cell production system according to any one of claims 1 to 16, further comprising a packaging device for packaging the somatic cells produced by the cell production device, wherein the housing houses the packaging device. A tubular member,
A liquid permeation filter disposed inside the tubular member;
A solution displacer comprising
In the cylindrical member,
A somatic cell introduction hole for introducing the solution containing the somatic cell prepared by the cell preparation device onto the liquid permeable filter;
A substitution solution inlet for introducing a substitution solution on the liquid permeation filter;
A somatic cell effusion pore for effluxing a replacement solution containing the somatic cell onto the liquid permeable filter;
Waste liquid outlet holes through which the solution permeating the liquid permeation filter flows out;
The system for producing somatic cells according to any one of claims 1 to 17, further comprising a solution replacer provided with   The waste liquid flow path connected to the waste liquid outflow port is further provided, and the flow of the solution in the waste liquid flow path is allowed when the solution of the solution containing the somatic cells is discarded, the somatic cells in the substitution solution The system for producing somatic cells according to claim 18, wherein the flow of the solution in the waste liquid delivery channel is not allowed when dispersing the   The somatic cell production system according to claim 18 or 19, wherein the replacement solution is a cryopreservation solution.   The somatic cell production according to claim 1, further comprising: a separation device for separating the pre-introduced cells from blood, wherein a solution containing the pre-introduced cells separated by the separation device passes through the pre-introduced cell delivery channel. system.