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WO2016043201A1 - Procédé de préparation d'une feuille de cellules activées de manière fonctionnelle par un traitement à faible teneur en oxygène - Google Patents

Procédé de préparation d'une feuille de cellules activées de manière fonctionnelle par un traitement à faible teneur en oxygène Download PDF

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WO2016043201A1
WO2016043201A1 PCT/JP2015/076207 JP2015076207W WO2016043201A1 WO 2016043201 A1 WO2016043201 A1 WO 2016043201A1 JP 2015076207 W JP2015076207 W JP 2015076207W WO 2016043201 A1 WO2016043201 A1 WO 2016043201A1
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sheet
cell
cdc
cell sheet
cells
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濱野公一
細山徹
上野耕司
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Yamaguchi University NUC
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Yamaguchi University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses

Definitions

  • the present invention relates to a method for producing a cell sheet functionally activated by low oxygen treatment, and a cell sheet produced by the production method.
  • Non-Patent Document 1 As Japan faces an aging society, the number of patients with heart failure in Japan, including potential patients, is approximately 1.6 million, and among them, the proportion of patients with ischemic heart disease including myocardial infarction is approximately half, 800,000 (Non-Patent Document 1).
  • old myocardial infarction caused by chronic cardiovascular infarction results in severe heart failure (chronic heart failure) with few subjective symptoms and extensive myocardial necrosis and thinning and scarring of the heart. Therefore, early establishment of an effective treatment method is desired.
  • Non-patent Document 2 mesenchymal stem cells and cardiosphere-derived cells (CDC) are considered to be promising cell types in the treatment of heart failure because they produce various blood vessel growth factors (such as VEGF) and can induce angiogenesis and cardiac function recovery after transplantation.
  • Non-patent Document 3 It has been pointed out that such transplanted cells have a low engraftment rate against infarcted hearts and insufficient ability to produce blood vessel growth factors. It was necessary to establish a methodology to overcome the challenges and maximize the characteristics of transplanted cells.
  • the cell sheet is capable of fixing a large amount of desired cells at the damaged site, and also enables transplantation of a moderately organized cell population according to the characteristics of the recipient tissue. It is a useful therapeutic material.
  • Non-Patent Documents 4 and 5 The cell sheet has been studied as a technique for improving the cell survival rate at the target site even in the field of circulatory system surgery, and particularly in the heart field, it has been reported that high engraftment in the infarcted heart and the accompanying promotion of myocardial regeneration are reported (Non-Patent Documents 4 and 5).
  • the present invention provides a method for producing a more effective cell sheet for use in treating damage to living tissue.
  • the present invention provides a method for producing a cell sheet that realizes sufficient functional improvement of the infarcted heart by transplanting the cell sheet in an infarcted heart in myocardial infarction.
  • the inventors once cultured cells on a culture substrate to form a cell sheet, and then the sheet was subjected to low temperature and low oxygen conditions.
  • the cell sheet obtained by culturing for a predetermined period (hypoxic preconditioning) and then peeling the sheet from the culture substrate plays an important role in angiogenesis compared to the cell sheet not subjected to the preconditioning treatment. It was found that the production amount of blood vessel growth factor was greatly increased.
  • the present invention was completed by finding improvements in the output rate and the left chamber diameter shortening rate.
  • the present invention includes the following (1) to (6).
  • a method for producing a cell sheet comprising the following steps (a) to (c): (A) culturing cells on a culture substrate to form a cell sheet derived from the cells; (B) culturing the cell sheet at a predetermined temperature and low oxygen condition for a predetermined period; (C) Step of peeling the cell sheet from the culture substrate after culturing under the conditions (2)
  • the method according to (1), wherein the cells are Cardiosphere-derived cells.
  • (6) A cell sheet produced by the method according to any one of (1) to (5).
  • the production amount of blood vessel growth factor that plays an important role in angiogenesis is greatly increased, and this is transplanted into a myocardial infarction model mouse, so that significant cardiac function (left ventricular ejection fraction) can be obtained.
  • a cell sheet capable of improving the left chamber inner diameter shortening rate can be obtained as a biological material for transplantation that can be used for treatment of ischemic heart disease typified by myocardial infarction.
  • CDC cardiosphere-derived cell
  • mVEGF vascular endothelial growth factor
  • mHGF hepatocyte growth factor
  • hVEGF vascular endothelial growth factor
  • B Number of blood vessel-like tube structures formed per field in the Normo group and the Hypo group. Enhancement of Akt signaling pathway in CDC sheets by hypoxic preconditioning. Increase in phosphorylated Akt in CDC sheets by hypoxic preconditioning stimulation (Hypo) versus normal culture (Normo). Addition of PI3K inhibitor LY294002 reduces phosphorylated Akt in a CDC sheet subjected to hypoxic preconditioning treatment (Hypoxia or Hypo) (A) and suppresses increased production of human VEGF (hVEGF) in the culture supernatant (B ). All remain at the level of normal culture (Normoxia or Normo).
  • A Schedule of treatment of human CDC sheet culture supernatant for mouse myofibroblast cell line SmcMF and immunofluorescence staining for phosphorylated histone H3 (pHH3).
  • B Image of immunofluorescence and DAPI staining of SmcMF treated with normal culture (Normo) or hypoxic preconditioning (Hypo) CDC sheet culture supernatant for pHH3.
  • C Proliferating myofibroblast ratio (pHH3-positive cells / total number of cells (%) per visual field) in SmcMF treated with Normo or Hypo-treated CDC sheet culture supernatant.
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular diameter shortening rate
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular diameter shortening rate
  • A Four weeks after transplantation of a CDC sheet subjected to normal culture (Normo) and hypoxic preconditioning treatment (Hypo) in a transplanted group to a mouse myocardial infarction model infarcted heart and a non-transplanted group (control group: Control) Measurement of left ventricular anterior wall thickness (thickness of infarcted myocardium).
  • Control group Control; CDC sheet non-transplanted group), Normo or Hypo-treated CDC sheet transplanted group (respectively Normo, respectively) at the border (border zone) between the infarcted part (IFA: Infarcted Area) and the non-infarcted part
  • IFA Infarcted Area
  • the present invention is a method for producing a cell sheet comprising the following steps (a) to (c). (A) culturing cells on a culture substrate to form a cell sheet derived from the cells; (B) culturing the cell sheet at a predetermined temperature and low oxygen condition for a predetermined period; (C) Step of peeling the cell sheet from the culture substrate after culturing under the conditions
  • the “cell sheet” is a general term for a culture of cells in which cells are bound in a sheet form, The cell sheet may be composed of one cell layer or may be composed of two or more cell layers.
  • any method may be used as long as it is possible to form a cell sheet derived from the cell by culturing the cell on a culture substrate, and suitable for each cell used. It can be performed under conditions ordinarily practiced in the technical field.
  • the culture temperature is 30 to 40 ° C., preferably 36 to 38 ° C.
  • the CO 2 concentration is 0 to 10%, preferably 4 to 6%
  • the O 2 concentration is atmospheric oxygen concentration (approximately 20%).
  • the conditions are not limited, and the culture temperature, CO 2 concentration, and O 2 concentration can be appropriately selected according to the characteristics of the cells to be cultured.
  • the culture temperature is 37 ° C.
  • the CO 2 concentration is 5%
  • the O 2 concentration is atmospheric oxygen concentration (approximately 20%).
  • the culture time is not particularly limited as long as it is a time necessary for forming a desired cell sheet.
  • the culture time may be about 10 hours to 240 hours, and preferably 12 hours to 168.
  • the culture time is about an hour, and in particular for a CDC sheet, it is not particularly limited, but it may be 48 to 96 hours.
  • the cell density initially seeded to form a cell sheet is not particularly limited as long as it is a condition normally performed in cell culture, but in order to produce a cell sheet in good condition, It is preferable that the cells are in a substantially confluent state at the time of seeding, for example, in a range of about 2 ⁇ 10 5 cells / cm 2 to 3 ⁇ 10 5 cells / cm 2 .
  • the state of the cells after the formation of the cell sheet is not particularly limited as long as it is in a healthy state, but may preferably be in a confluent state.
  • the “culture substrate” may be any cell as long as cells can form a cell sheet on the surface thereof, and at least includes a flat portion to which cells can adhere, Typically, it is a cell culture dish or a cell culture bottle (or flask), and a commercially available culture dish or the like can be used, and the material is not particularly limited.
  • the culture substrate material include polyethylene, polypropylene, polyethylene terephthalate, and the like.
  • the culture surface of the “culture substrate” is made of a material whose physical properties change due to a temperature change or the like (temperature responsive material), or the culture surface of the culture substrate is layered by the temperature responsive material. It may be coated.
  • a cell adhesion component and / or a cell adhesion inhibitory component may be present on the culture surface of the main culture substrate.
  • the cell adhesion component may be any component that is usually used for adhering cells to the culture surface in cell culture technology, such as collagen, fibronectin, laminin, heparan sulfate proteoglycan, cadherin, gelatin, Examples include fibrinogen, fibrin, poly L lysine, hyaluronic acid, platelet-rich plasma, and polyvinyl alcohol.
  • the cell adhesion-inhibiting component may be any component that is usually used for inhibiting cell adhesion to the culture surface in the cell culture technique, and examples thereof include albumin and globulin.
  • any animal species or tissue-derived cell can be used.
  • examples of such cells include, but are not limited to, myoblasts, cardiomyocytes, cardiosphere-derived cells (CDC), mesenchymal stem cells, fibroblasts, hematopoietic stem cells, intestinal stem cells, hair follicle stem cells, Breast stem cells, neural stem cells, endothelial stem cells, olfactory mucosal stem cells, embryonic stem cells, iPS cells, synovial cells, epithelial cells (eg, corneal epithelial cells, oral mucosal epithelial cells), endothelial cells, hepatocytes, pancreatic cells, periodontal ligament Examples include cells and skin cells, and preferred are mesenchymal stem cells or CDC.
  • cardiosphere related to CDC is a three-dimensional cell mass obtained by culturing a myocardial biopsy specimen separated into small pieces and cultivating proliferating cells generated around the specimen, and dispersing the cell mass. Later, the recovered cells are CDC.
  • CDC is a tissue stem cell that can be isolated from a small amount of heart tissue and has a very high myocardial differentiation efficiency. Therefore, CDC is suitable as a biological material for transplantation that can be used for the treatment of ischemic heart disease represented by myocardial infarction. is there.
  • the “cell” used in the formation of the cell sheet of the present invention includes a step of isolating a desired cell from a tissue or biological fluid collected from a subject, a step of growing the isolated cell, It may be produced through a process of differentiation into a cell line or a commercially available cell line already established. Further, only one type of cell may be used for forming the present cell sheet, but two or more types of cells may be used.
  • a medium suitable for the cells to be cultured can be appropriately selected and used.
  • MEM, DMEM, F12, IMEM, IMDM, RPMI-1640, Neurobasal, etc. can be mentioned as media that can be generally used. You may purchase and use these culture media. Moreover, these culture media may be used independently or may be used in combination of 2 or more types. Furthermore, you may use for a culture medium, adding an appropriate additive as needed.
  • L-type amino acids for example, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-trionine, L-tryptophan, L-tyrosine, etc.
  • vitamins eg, folic acid, riboflavin, thiamine, etc.
  • D-glucose and other animal sera such as fetal bovine serum, horse serum, etc. But you can.
  • a buffering agent for example, PBS, HEPES, MES, HANK'S etc.
  • a cell growth factor or the like may be added as appropriate according to the characteristics of the cells to be cultured.
  • the medium used when CDC is cultured and formed into a cell sheet may be IMDM, and fetal calf serum (10%) and L-glutamine (1 mM) may be used as additives.
  • the step (b) is a step of culturing the above cell sheet for a predetermined period at a predetermined temperature and low oxygen condition.
  • the low oxygen condition is an oxygen concentration condition lower than the atmospheric oxygen concentration (approximately 20%).
  • the predetermined temperature, low oxygen condition, and treatment period vary depending on the cell from which the cell sheet is derived, and appropriate conditions for each cell sheet are determined by methods that can be easily studied by those skilled in the art, such as preliminary experiments. Can be determined.
  • the suspension of fibroblasts when the O 2 concentration 0, 1, 2, 3, or 5% and the hypoxic preconditioning treatment was performed, the O 2 concentration 0,1,2 It has been confirmed that any of 3% or 5% shows equivalent VEGF production.
  • the hypoxic condition is that the O 2 concentration is 0% to 8%, preferably 0.1% to 5%, Preferably it is 2%.
  • the culture temperature is 30 ° C. to 36 ° C., preferably 32 ° C. to 34 ° C., more preferably 33 ° C.
  • the treatment period is, for example, 12 hours to 72 hours, preferably 24 hours.
  • the cell sheet may be immediately transferred to the step (c) or may be returned to the normal culture condition for a certain period and then transferred to the step (c).
  • the conditions can be set for each cell sheet, and the fixed period when returning to normal culture conditions for a fixed period is not particularly limited. For example, in the CDC sheet, it is 1 to 12 hours. It may be 1 hour to 6 hours, more preferably 1 hour to 2 hours.
  • the step (c) is a step of peeling the cell sheet from the culture substrate after culturing under the above conditions. Peeling of the cell sheet from the culture substrate can be carried out by a method that does not damage the sheet-like structure.
  • the sheet-like cell culture is directly picked with tweezers and peeled off from the culture surface, or Alternatively, a physical technique such as peeling the cells from the culture surface by pipetting may be used.
  • an enzyme treatment such as trypsin or collagenase may be performed, and an appropriate method can be selected according to the properties of the cells.
  • the cell sheet can be peeled and collected by covering the upper surface of the cell sheet with a substrate having affinity for cells, such as a PVDF membrane or a nitrocellulose membrane, and copying the cells onto the membrane.
  • a substrate having affinity for cells such as a PVDF membrane or a nitrocellulose membrane
  • the above-described cell detachment and recovery may be performed after the temperature of the container is lowered to, for example, about 0 to 30 ° C. Good.
  • the present invention includes a cell sheet produced by the method of the present invention. Since the function of the cell sheet is enhanced by culturing for a predetermined period under low temperature and low oxygen conditions, an excellent therapeutic effect as a biological material for transplantation can be expected. That is, the cell sheet produced according to the present invention can be used as a biological material for transplantation into any tissue, organ, organ, etc., for the purpose of improving the malfunction of the animal. .
  • the “animal” is not particularly limited, but is preferably an animal whose function is expected to be improved by transplantation. Specifically, in addition to humans, pet animals such as dogs, cats and rabbits, cattle , Livestock animals such as pigs, sheep and horses.
  • tissue, organ, organ and the like to which the cell sheet produced according to the present invention is transplanted are not particularly limited, but heart, brain, lung, kidney, liver, pancreas, small intestine, bone marrow, cornea, skin, skeletal muscle
  • the heart is preferable.
  • the disease in the case of targeting the heart is not particularly limited, but examples include ischemic heart disease represented by angina pectoris and myocardial infarction, dilated cardiomyopathy, etc., preferably myocardial infarction, more preferably This is a so-called old myocardial infarction (OMI) that has passed 30 days or more since the onset of myocardial infarction.
  • OMI old myocardial infarction
  • the present invention also includes a method for treating or preventing a disease using the cell sheet produced by the method of the present invention.
  • the target disease is not particularly limited as long as it can be treated or prevented by transplanting the cell sheet.
  • Examples include blood heart disease and dilated cardiomyopathy, preferably myocardial infarction, more preferably old myocardial infarction.
  • a cell sheet is formed from mesenchymal stem cells or CDC. I can do it.
  • mesenchymal stem cells and CDC are collected from a patient suffering from the disease and used to form an autologous stem cell sheet, the survival rate of transplanted cells in the heart is improved by suppressing rejection.
  • Example 1 Cardiosphere-derived cells (CDC) derived from adult hearts were prepared and formed into cell sheets.
  • a heart tissue piece was collected from the right atrium of the adult heart by biopsy, and was cut into about 0.5 mm square, and the heart tissue piece was allowed to stand on a fibronectin-coated culture dish.
  • Medium exchange IMDM containing 10% FBS / 1 mM L-glutamine
  • EDC heart tissue-derived cells
  • the composition of the cardiosphere formation medium is based on 35% IMDM and 65% DMEM / F12, 3.5% fetal bovine serum, 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1 unit / mL thrombin, 1% Consists of B-27, 80 ng / mL bFGF, 25 ng / mL EGF, 4 ng / mL Cardiotrophin-1.
  • a fine cardiosphere was formed, and the culture medium was changed for 48 hours and cultured for 96 hours.
  • the recovered cardiosphere was seeded on a culture dish coated with fibronectin, and again subjected to adhesion culture and cultured for one week.
  • a cell that has come out from the adhered cardiosphere is a cardiosphere-derived cell (cardiosphere-derived cell; CDC).
  • the medium used for obtaining CDC was IMDM containing 10% FBS / 1 mM L-glutamine, and the medium was changed once every two days.
  • the prepared adult heart-derived CDC expresses myocardial stem cell marker c-Kit and, after seeding in a culture dish, expresses vascular smooth muscle cell marker ( ⁇ SMA) and vascular endothelial cell marker (CD31) (Fig. 1) It was confirmed that it has the ability to differentiate into vascular smooth muscle cells and vascular endothelial cells. According to the above method, a CDC-derived cell sheet (CDC sheet) was formed.
  • Example 2 The CDC sheet was subjected to a treatment (low oxygen preconditioning) for culturing for a predetermined period at a predetermined temperature and low oxygen condition.
  • a CDC sheet prepared using a temperature-responsive culture dish (Cellseed) was cultured for 24 hours under low oxygen conditions of 33 ° C., O 2 concentration of 2%, and CO 2 concentration of 5%.
  • Example 3 In order to clarify the presence or absence of damage to the cell sheet by the hypoxic preconditioning, expression analysis of caspase 7 (Caspase 7), which is an index of apoptosis (cell death), was performed.
  • CDC sheet is 24 hours, O 2 concentration 2%, CO 2 concentration 5%, 33 ° C condition (low oxygen preconditioning condition) or 24 hours, O 2 concentration 20%, CO 2 concentration 5%, 37 ° C condition ( After culturing under normal culture conditions), it was dissolved in RIPA buffer and analyzed for caspase 7 protein expression by Western Blot method.
  • As a primary antibody an anti-Caspase 7 antibody (# 9492: diluted 1000 times) of Cell Signaling Technology was used.
  • Example 4 Confirmation of the effect of hypoxic preconditioning treatment on the cell sheet prepared from the mouse by the above method on the ability of the sheet to produce vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) Therefore, the amount of VEGF and HGF produced on the CDC sheet according to the presence or absence of the treatment was measured.
  • both VEGF and HGF were significant in the hypoxic preconditioning group (Hypo Sheet). Production was promoted (FIG. 3). In particular, a significant increase of 23 times or more was observed in VEGF.
  • Example 5 Whether cell sheets prepared from humans by the method according to Example 1 as well as mice were subjected to hypoxic preconditioning treatment was confirmed to enhance the VEGF production ability of the sheet.
  • the culture supernatant of a human CDC sheet that had been subjected to hypoxic preconditioning (cultured at 33 ° C., O 2 concentration 2%, CO 2 concentration 5% for 24 hours) was collected, and in the same manner as in Example 4, ELISA (R & D Systems) ),
  • the VEGF concentration secreted from the CDC sheet was measured and compared with the VEGF concentration in the treatment group under normal conditions (cultured at 37 ° C., O 2 concentration 20%, CO 2 concentration 5% for 24 hours). Similar to the mouse CDC sheet, VEGF increased more than 2-fold in the hypoxic preconditioning group (Hypo Sheet) compared to the group cultured under normal conditions (Normo Sheet) (FIG. 4).
  • Example 6 Whether hypoxic preconditioning induces an enhanced angiogenic effect of the cell sheet was verified by an in vitro experimental system using cultured human vascular endothelial cells. After culturing human CDC sheets for 24 hours under conditions of 33 ° C., O 2 concentration 2%, CO 2 concentration 5% or 37 ° C., O 2 concentration 20%, CO 2 concentration 5%, the culture supernatant is recovered. Then, it was verified whether or not angiogenesis was promoted by culturing human umbilical cord vascular endothelial cells (HUVEC: purchased from Lonza) with each culture supernatant.
  • HUVEC human umbilical cord vascular endothelial cells
  • HUVECs were cultured for 12 hours in the culture supernatant of the CDC sheet, and the number of formed blood vessel-like tube structures (FIG. 5A) per field was counted. Tube formation was significantly enhanced in the HUVEC under culture in the culture supernatant of the CDC sheet treated with hypoxia preconditioning, suggesting that the angiogenic factor expression of the cell sheet was increased by exposure to hypoxia (FIG. 5B). . Since VEGF and HGF expression of CDC sheet is enhanced by hypoxic preconditioning (Example 4 and Example 5), it is presumed that the increase in production of the vascular factor group affected the angiogenic ability of HUVEC.
  • Example 7 In order to clarify through which signal transduction pathway the increase in VEGF expression by the hypoxic preconditioning treatment to the CDC sheet shown in Example 4 was performed, pathway analysis was performed (FIG. 6 left figure). ). CDC sheets subjected to hypoxic preconditioning were dissolved in Radio-Immunoprecipitation Assay (RIPA) buffer and phosphorylated states of 43 types of kinase proteins using Human Phospho-Kinase Antibody Array (registered trademark) of R & D Systems. Comparison (pathway analysis). The assay was performed according to the manual attached to this kit, and the density of each kinase protein spot immobilized on the membrane was digitized with ImageJ software and then compared with a group cultured under normal conditions.
  • RIPA Radio-Immunoprecipitation Assay
  • the Akt signaling pathway is known as a direct regulator of VFGF, it is assumed that the increase in VEGF expression generated in the CDC sheet by hypoxic preconditioning was caused by the enhancement of the Akt signaling pathway. Is done.
  • Example 8 In Example 7, it was shown that hypoxic preconditioning stimulation activates multiple signal transduction pathways in the CDC sheet. Focusing on the signal transduction pathway via Akt, it was verified whether or not it actually functions as a mediator of hypoxic preconditioning stimulation.
  • Human CDC sheets are cultured for 24 hours under conditions of 33 ° C., O 2 concentration 2%, CO 2 concentration 5% (low oxygen condition) or 37 ° C., O 2 concentration 20%, CO 2 concentration 5% (normal conditions). Then, when dissolved in RIPA buffer and phosphorylated Akt (pAkt) expression was compared by Western Blot method, increased phosphorylation of Akt occurred in the hypoxic condition group (Hypo) compared to the normal condition group (Normo). (Fig. 7).
  • Example 9 Furthermore, the human CDC sheet was added to the above hypoxic preconditioning conditions (Hypoxia or Hypo) in a state where an inhibitor of Akt phosphorylation regulator PI3K (LY294002; Cell Signaling Technology: # 9901: 10 microM) was added to the medium. After exposure, cells and culture supernatant were collected, and phosphorylated Akt expression and VEGF production were confirmed. In the LY294002 addition group, both Akt phosphorylation and VEGF production enhancement by hypoxic preconditioning stimulation were suppressed, and normal culture was performed. Stayed at the condition (Noroxia or Normo) level (FIGS. 8A and B). This result suggests that hypoxic preconditioning promotes VEGF production enhancement of the CDC sheet via the PI3K / Akt signaling pathway.
  • hypoxic preconditioning promotes VEGF production enhancement of the CDC sheet via the PI3K / Akt signaling pathway.
  • Example 10 The myofibroblast proliferation inhibitory effect of the CDC sheet by hypoxic preconditioning was verified using a cultured cell line.
  • the myofibroblast cell line SmcMF Korean et al., World J Gastroenterol. 19: 2629-2737
  • FBS / DMEM 10% FBS / DMEM (35,000 cells / well).
  • the culture supernatant collected from each time-cultured CDC sheet was replaced with the 10% FBS / DMEM, and SmcMF was further cultured for 24 hours.
  • the cultured cells were subjected to immunofluorescence staining and DAPI staining with anti-phosphorylated histone H3 antibody (anti-pHH3 antibody; Cell Signaling Technology: # 9701: diluted 200-fold) after fixation with 4% paraformaldehyde.
  • the number of SmcMF cells was counted (FIG. 9A). Specifically, “the number of anti-pHH3 antibody positive cells / the number of DAPI positive cells ⁇ 100” was calculated from the fluorescence-stained image (FIG.
  • Endoglin is known as one of the factors having an inhibitory effect on myofibroblasts. Then, when the expression level of endoglin was changed in the CDC sheet subjected to the treatment by the hypoxic preconditioning treatment for the CDC sheet, it was confirmed that increased endoglin expression occurred (FIG. 10). . Specifically, under low oxygen conditions (Hypoxia; 33 ° C., O 2 concentration 2%, CO 2 concentration 5%) or normal conditions (Normoxia; 37 ° C., O 2 concentration 20%, CO 2 concentration 5%) After processing the CDC sheet for a time, a cell extract was prepared from the CDC sheet. Thereafter, Western blotting was performed for endoglin and actin expressed in the extract (FIG.
  • Example 12 In order to clarify whether cardiac function is improved by transplanting the CDC sheet, first, a mouse old heart failure model (oMI) was created, and the left ventricle 4 weeks after ligation of the left anterior descending coronary artery (LAD) Ejection rate (LVEF) and left ventricular diameter shortening rate (LVFS) were measured. After thoracotomy of mice (C57BL / 6; male; 10 weeks old) under anesthesia, the left anterior descending coronary artery was ligated with 8-0 polypropylene suture, and the left ventricular ejection fraction was about 30 in echocardiography after 30 days. % (Normal value is approximately 70%) was used as a mouse oMI model for transplantation experiments.
  • LAD left anterior descending coronary artery
  • LVEF left anterior descending coronary artery
  • LVFS left ventricular diameter shortening rate
  • LVEF (%) is (left ventricular end-diastolic volume-left ventricular end-systolic volume) ⁇ left ventricular end-diastolic volume ⁇ 100
  • LVFS (%) is (left ventricular end-diastolic diameter-left ventricular end-systolic diameter) ⁇ left
  • the calculation was performed with the end-diastolic chamber diameter ⁇ 100.
  • the left ventricular volume and the left ventricular diameter were measured by M mode at the time of echocardiography measurement. It was confirmed that the numerical values of LVEF and LVFS in the oMI group were significantly lower than those in the sham operation group (Sham Operation group; Sham) (FIG. 11).
  • Example 13 For the mouse CDC sheet, a group to be subjected to a hypoxic preconditioning treatment and a group not to be performed (normal culture group) 24 hours before transplantation were prepared, and the CDC sheet group was used as the mouse old heart failure model (oMI). Transplantation was performed to examine differences in LVEF and LVFS between the two groups. After thoracotomy of mice (C57BL / 6; male; 10 weeks old) under anesthesia, the left anterior descending coronary artery was ligated with 8-0 polypropylene suture, and the left ventricular ejection fraction was about 30 in echocardiography after 30 days. % (Normal value is approximately 70%) was used as a mouse oMI model for transplantation experiments.
  • oMI mouse old heart failure model
  • LVEF (%) is (left ventricular end-diastolic volume-left ventricular end-systolic volume) ⁇ left ventricular end-diastolic volume ⁇ 100
  • LVFS (%) is (left ventricular end-diastolic diameter-left ventricular end-systolic diameter) ⁇ left
  • the calculation was performed with the end-diastolic chamber diameter ⁇ 100.
  • the left ventricular volume and the left ventricular diameter were measured by M mode at the time of echocardiography measurement.
  • hypoxic preconditioning treatment and the untreated CDC sheet were transplanted into oMI, LVEF and LVFS were measured after 4 weeks, and the recovery rates ( ⁇ LVEF and ⁇ LVFS) from before each transplantation were compared (left figure in FIG. 12: ⁇ LVEF). , Right figure: ⁇ LVFS), all showed a significant increase in recovery rate in the hypoxic preconditioning treatment group.
  • significant cardiac function recovery was confirmed in the treated CDC sheet as compared with the CDC sheet not treated with hypoxic preconditioning, and the function enhancement of CDC sheet (expression of VEGF and HGF) by the hypoxic preconditioning treatment was confirmed. (Increase) was suggested to be extremely effective in treating chronic ischemic heart disease.
  • Example 14 Although it was shown in Example 13 that the cardiac function of the failing heart was recovered by transplanting the CDC sheet, it is not clear by what mechanism the cardiac function was improved. Therefore, histological analysis was performed on failing hearts transplanted with sheets. Hypoxic preconditioning treatment (33 ° C, O 2 concentration 2%, CO 2 concentration 5% culture for 24 hours) and normal culture (37 ° C, O 2 concentration 20%, CO 2 concentration 5% culture for 24 hours) The left ventricular anterior wall thickness (infarct) was compared 4 weeks after the transplantation, and the cell wall non-transplanted group was compared with the anterior wall in the cell sheet transplanted group (normal culture group). A significant thickening of thickness was observed (FIG. 13A).
  • hypoxic preconditioning treatment group Although there was no significant difference between the hypoxic preconditioning treatment group and the normal culture group, a tendency of higher values was observed in the hypoxic preconditioning treatment group (FIG. 13A). This suggests that remodeling of the infarcted heart is caused by cell sheet transplantation, particularly by treatment with hypoxic preconditioning.
  • the anterior wall thickness of the left ventricle was calculated using an ultrasonic high-resolution imaging system Vevo 770 (manufactured by VisualSonics) used for echo measurement.
  • vascular endothelial cells Lectin; Vector Laboratories: DL-1174: diluted 200-fold
  • cardiomyocytes Total area of vascular endothelial cells in border zone (%)” after immunofluorescent staining for (cTnT: Abcam; ab10214: diluted 100 times) (ratio of total area occupied by lectin-positive vascular endothelial cells in one border zone visual field) BZ-II analyzer (manufactured by Keyence Co., Ltd.) was used as an index, and the CDC cell sheet transplanted group subjected to hypoxic preconditioning was significantly different from the normal cultured CDC cell sheet transplanted group. Increase was observed (FIG. 13B). That is, this event suggests that the CDC cell sheet treated with hypoxia preconditioning promotes angiogenesis in the infarcted heart.
  • infarct area in the failing heart was visualized by Masson trichrome staining, normal heart (Sham group), infarcted heart (Control group), infarcted heart transplanted with a normal cultured CDC sheet (Normo group), hypoxic pre-treatment Between the infarcted heart (Hypo group) transplanted with the conditioned CDC sheet, “Infarcted Area; Infarct Area (%)” (calculate the infarct area / total cross-sectional area of the heart using the BZ-II analyzer). When compared, a significant reduction in infarct area was observed in the Hypo group (FIG. 13C). This suggests that the hypoxic preconditioning promoted the scar reduction effect of the CDC sheet, and the thickening of the left ventricular anterior wall thickness shown above was caused by increased neovascularization and myocardial remodeling due to scar reduction. Infer what happened.
  • HIF-1alpha is known as a major sensing molecule for oxygen concentration, and is usually decomposed immediately after synthesis under oxygen concentration, but under low oxygen conditions, decomposition is suppressed and a downstream hypoxia responsive molecule group is activated.
  • VEGF which is an angiogenic factor, is known as a main downstream target molecule of HIF-1alpha. Therefore, in order to confirm the hypoxic responsiveness of the CDC sheet, Western blot analysis for HIF-1alpha expression was performed. A human CDC sheet was obtained under conditions of 33 ° C., O 2 concentration 2%, CO 2 concentration 5% (low oxygen preconditioning treatment) or 37 ° C., O 2 concentration 20%, CO 2 concentration 5% (normal culture).
  • HIF-1alpha was detected only in the CDC sheet (Hypo) treated with hypoxia, and it was recognized in the CDC sheet (Normo) in normal culture. I could't. From this, it was confirmed that a hypoxic response occurred only in the former (FIG. 14).
  • an anti-HIF-1 alpha antibody (# 3716: diluted 1000 times) of Cell Signaling Technology was used.
  • Example 16 The presence or absence of extracellular matrix-degrading enzyme matrix metalloproteinase 2 (MMP-2) and / or matrix metalloproteinase 3 (MMP-3) in the culture supernatant of human CDC sheet was detected by ELISA. .
  • MMP-2 matrix metalloproteinase 2
  • MMP3G0 Quantikine MMP-3 immunoassay kit
  • DMP2F0 Quantikine Human MMP-2 immunoassay kit manufactured by R & D systems were used.
  • MMP-3 was not expressed, and it was revealed that MMP-2 was specifically expressed (FIG. 15). This suggested that the degradation enzyme may have a function of digesting scar tissue formed in the infarcted heart in the CDC sheet.
  • the present invention provides a method for producing a cell sheet having an enhanced function by performing a hypoxic preconditioning treatment, and thus has high utility in the medical field related to transplantation, and in particular, treated with hypoxic preconditioning.
  • Mesenchymal stem cells and CDC-derived cell sheets are extremely effective in treating chronic ischemic heart disease and contribute to the development of the medical field.

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Abstract

La présente invention concerne un procédé de préparation d'une feuille de cellules qui est utilisée de manière plus utile dans le traitement de lésions dans des tissus vivants. La présente invention concerne en particulier un procédé de préparation d'une feuille de cellules qui permet d'obtenir une amélioration fonctionnelle suffisante d'un myocarde infarci par la transplantation de ladite feuille de cellules destinée à un myocarde infarci dans des cas d'infarctus du myocarde. Plus particulièrement, la présente invention concerne un procédé de préparation d'une feuille de cellules, le procédé comprenant : une étape consistant à mettre en culture des cellules sur un substrat de culture pour former une feuille de cellules dérivées desdites cellules ; une étape consistant à mettre en culture la feuille de cellules dans des conditions à faible teneur en oxygène, à une température prédéfinie, pendant une période de temps prédéfinie ; et une étape consistant à détacher la feuille de cellules du substrat de culture après ladite mise en culture dans lesdites conditions.
PCT/JP2015/076207 2014-09-16 2015-09-16 Procédé de préparation d'une feuille de cellules activées de manière fonctionnelle par un traitement à faible teneur en oxygène Ceased WO2016043201A1 (fr)

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CN108379662A (zh) * 2018-02-08 2018-08-10 深圳大图科创技术开发有限公司 一种干细胞在心脏移植模型的应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021509291A (ja) * 2017-12-27 2021-03-25 玄 鈴木 カーディオスフェア由来細胞シート、ならびにそれを製造する方法および使用
JP7313375B2 (ja) 2017-12-27 2023-07-24 玄 鈴木 カーディオスフェア由来細胞シート、ならびにそれを製造する方法および使用
KR20250150543A (ko) 2023-02-14 2025-10-20 고쿠리츠다이가쿠호우진 야마구치 다이가쿠 간엽계 세포를 포함하는 세포 집단, 세포 집단을 포함하는 의약 조성물, 세포 집단으로부터 얻어진 엑소좀 및 세포 집단을 제작하는 방법

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