WO2016190704A1 - Cellules adhérentes postnatales améliorées et procédé de préparation associé - Google Patents

Cellules adhérentes postnatales améliorées et procédé de préparation associé Download PDF

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WO2016190704A1
WO2016190704A1 PCT/KR2016/005636 KR2016005636W WO2016190704A1 WO 2016190704 A1 WO2016190704 A1 WO 2016190704A1 KR 2016005636 W KR2016005636 W KR 2016005636W WO 2016190704 A1 WO2016190704 A1 WO 2016190704A1
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adherent cells
cells
cell
enzyme
postpartum
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Korean (ko)
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김혜선
강아름
김현주
박경미
주상언
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주식회사 차바이오텍
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Priority to US15/577,633 priority Critical patent/US10479978B2/en
Priority claimed from KR1020160065593A external-priority patent/KR20160140492A/ko
Publication of WO2016190704A1 publication Critical patent/WO2016190704A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

Definitions

  • Cell therapy is a medicine used for the purpose of preventing or treating certain diseases by changing the characteristics of cells by proliferating or selecting cells in vitro to restore the function of cells and tissues.
  • the field is receiving.
  • Mesenchymal stem cells are pluripotent stem cells that can proliferate and differentiate into various lineages, also called mesenchymal progenitor cells.
  • Mesenchymal stem cells can be differentiated into bone, fat, cartilage, nerve, muscle, and bone marrow stromal cells, etc., depending on conditions, and thus have various therapeutic effects.
  • Mesenchymal stem cells are a kind of adult stem cells, and can be separated with hematopoietic stem cells, which are mainly separated from bone marrow.
  • mesenchymal stem cells having a characteristic of being cultured in a suspended state, they are attached to a culture dish. Have Mesenchymal stem cells isolated and cultured under these culture conditions have been used in various experiments and / or clinical trials. However, mesenchymal stem cells have been studied mainly by separating them from bone marrow, fat, and umbilical cord blood, and the donor suffers from the collection and separation of bone marrow-derived mesenchymal stem cells, and the separation efficiency of mesenchymal stem cells is low. There is this.
  • Placenta is a tissue that is discarded after childbirth and is easy to collect and contains many types of adherent cells.
  • the mesenchymal cells, decidual cells, trophoblasts, amnion, and epithelium are classified by site. There are various cells, such as endothelial cells. Zhang et al. Have disclosed a method for isolating mesenchymal progenitor cells from the placenta and the characteristics of the resulting mesenchymal progenitor cells (Experimental Hematology 32 (2004) 657-664).
  • the amniotic sac and the decidual membrane were removed from the placenta, washed with phosphate buffer, and the perfusion and culture fluids were flowed through the arterial-vein circuit to remove residual blood from the tissue. Subsequently, the culture medium is soaked for 12 to 24 hours, monocytes are obtained by a percol density gradient, and then resuspended in a medium containing fetal bovine serum to separate mesenchymal progenitor cells.
  • the method may be a laboratory application because it must perform a complex step and undergo a process such as a percol density gradient, but because of culturing the mesenchymal progenitor cells in the placenta itself for a long time, it will result in a mixture of monocytes present in the placenta.
  • there are many difficulties in clinical application since it is difficult to stably separate and purify healthy uniform cells stably.
  • the method has a problem that the purity of the resulting cell is reduced by mixing with other cells in which the obtained cells are separated throughout the placental villi by using the placenta from which the amniotic cavity and the decidual membrane are removed.
  • the stable supply of sufficient cells and sufficient cell numbers to be effective enough to be used as a therapeutic agent should be an important part of cell therapy research.
  • research on a method of manufacturing a cell population capable of treatment and regeneration is urgent.
  • the conventional separation method of placental-derived cells has a disadvantage in that the separation efficiency is low, and it is a new adherent type that can be used as a cell therapy while simultaneously satisfying the proliferative and differentiating ability of placental-derived cells. There is a need for a method for isolating cells.
  • One aspect includes obtaining placental derived tissue from the separated placenta; Collecting a cell population by adding an enzyme mixture solution to the placental tissue; Attaching and culturing the harvested cell population to a container, and then treating the animal component free (ACF) recombinant enzyme to separate postpartum adherent cells; And passaging the isolated postpartum adherent cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin under low oxygen conditions compared to 21% of normal oxygen conditions. It is to provide a method of manufacturing.
  • ACF animal component free
  • Another aspect includes adding a solution of an enzyme mixture to isolated placental derived tissues to harvest cell populations; Attaching and culturing the collected cell population to a flask, and then treating the animal component free (ACF) recombinant enzyme to separate postpartum adherent cells; And passaging the isolated postpartum adherent cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin under low oxygen conditions compared to the normal oxygen condition of 21%. It is to provide a method for increasing the manufacturing efficiency.
  • ACF animal component free
  • One aspect provides a method of making enhanced postpartum adherent cells that separates and cultures enhanced postpartum adherent cells (ePACs) from isolated placental placental tissue.
  • ePACs enhanced postpartum adherent cells
  • the improved postpartum adherent cell production method includes the steps of obtaining placental derived tissue from the separated placenta; Collecting a cell population by adding an enzyme mixture solution to the placental tissue; Attaching and culturing the harvested cell population to a container, and then treating the animal component free (ACF) recombinant enzyme to separate enhanced postpartum adherent cells; And passaging the isolated enhanced postpartum adherent cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin at low oxygen conditions compared to normal oxygen conditions of 21%.
  • FGF-4 fibroblast growth factor-4
  • placenta refers to an organ made for the fetus during pregnancy of a mammal, and in one embodiment, may be the human placenta.
  • One side of the placenta is in contact with the mother and the other is in contact with the fetus, and the space between the mother's blood contains the nourishment to the fetus.
  • the placenta consists of three layers of amnion, chorion, and decidual membrane, and includes the umbilical cord.
  • Amniotic membrane is a thin, transparent membrane that surrounds the fetus and contains amniotic fluid.
  • the decidual membrane is a membrane formed by deforming epithelial cells of the uterus so that the fertilized egg implants in the uterus.
  • the chorion is the membrane between the amnion surrounding the fetus or amniotic fluid and the decidual membrane. It occurs in the fertilized egg and forms part of the egg.
  • the umbilical cord connects the fetus with the placenta, where the material exchange between the mother and fetus takes place, and through the fetus's navel to the heart.
  • the term "enhanced Postnatal adherent cells (ePACs)” has the shape of fibroblasts and is capable of infinite proliferation and the ability to differentiate into cell lines such as fat, osteocytes, and chondrocytes. It can mean a cell having. It may also mean a cell that is not derived from the inner cell mass of the blastocyst.
  • Postpartum adherent cells from a human placenta isolated in vitro are, for example, (1) amnion epithelial cells, (2) adherent cells derived from amnion, (3) chorionic adherent cells, (4) chorionic trophoblast cells, And (5) umbilical cord cells.
  • the placenta may use a placenta that is separated and discarded after delivery from a healthy mother. That is, the term "isolated placenta" refers to the placenta that is separated after giving birth from the mother's mother.
  • the separated placenta can be stored in sterile containers and ice quickly after separation.
  • Obtaining placental tissue from the isolated placenta can be obtained by conventional anatomical methods, for example, by cutting the placental tissues present in the placenta into various sites with sterile scissors.
  • the resulting placental tissue is washed twice or more with phosphate buffered saline (PBS) containing antibiotics (eg, penicillin, streptomycin, gentamicin, etc.) to remove contaminants such as blood present in the tissue. can do.
  • PBS phosphate buffered saline
  • antibiotics eg, penicillin, streptomycin, gentamicin, etc.
  • the placental tissue may be amnion, chorionic or decidual tissue.
  • the amnion can be obtained by pulling the chorionic membrane off from the separated placenta, and then scraping off the chorion membrane to remove the chorion.
  • Placental tissue collected as described above may be directly enzymatically treated or further enzymatically treated using sterile scissors or the like.
  • the placental tissue may be finely cut (eg, about 5 mm or less) using sterile scissors or the like, and then the cut cells may be enzymatically treated.
  • the enzyme mixture solution may refer to an enzyme mixture obtained by mixing various enzymes, or a reaction enzyme solution, and the enzyme mixture solution reacting with the placental tissue may dissolve the tissue to separate adherent cells from the tissue.
  • the enzyme mixture solution may include collagenase, trypsin, and dispase, and the enzyme mixture solution may also include collagenase, trypsin, and Water, including saline, and saline, for example, HBSS (Hank's Balanced Salt Solution).
  • the collagenase may refer to an enzyme that breaks down the peptide bonds of collagen, and may include collagenase type I, type II, type III, type IV, or a combination thereof.
  • the enzyme mixture solution may further comprise a DNA hydrolase (DN) I or II.
  • the concentration of collagenase in the enzyme mixture solution may be, for example, 0.5 mg / ml to 5 mg / ml, 0.5 mg / ml to 3 mg / ml, 0.8 mg / ml to 2 mg / ml, or 0.8 mg / ml To 1.5 mg / ml, and in one embodiment, 1.2 mg / ml.
  • the concentration of trypsin in the enzyme mixture solution is for example 1 mg / ml to 5 mg / ml, 1 mg / ml to 3 mg / ml, 1.5 mg / ml to 2.5 mg / ml, or 1.5 mg / ml to 2 mg / ml, and in one embodiment, may be 1.8 mg / ml.
  • the concentration of dispase in the enzyme mixture solution may be, for example, 0.1 U / ml to 5 U / ml, 0.1 U / ml to 3 U / ml, 0.5 U / ml to 2.5 U / ml, or 0.5 U / ml To 1.5 U / ml, and in one embodiment, 1 U / ml.
  • the concentration of the DNA hydrolase in the enzyme mixture solution is, for example, 0.001 mg / ml to 1 mg / ml, 0.001 mg / ml to 0.5 mg / ml, 0.01 mg / ml to 0.25 mg / ml, or 0.01 mg / ml To 0.05 mg / ml, and in one embodiment may be 0.025 mg / ml.
  • the reaction between the tissue and the enzyme mixture solution may be carried out by performing shaking, wherein the shaking is about 20 to 40 °C, about 30 to 40 °C, or 35 to 40 °C, for example, It may be performed at 37 ° C., may be performed for about 5 to 60 minutes or 10 to 30 minutes, and may be performed twice, for example, for 10 to 30 minutes.
  • tissue cells for example, amniotic cells (ie adherent cells) from the enzyme reaction solution can be carried out by methods known in the art, for example, after centrifugation, Cell bodies can be used to separate cells.
  • ePACs enhanced postpartum adherent cells
  • separating the population of cells is used interchangeably with separating enhanced postpartum adherent cells, ie, separation of amnion cells is used interchangeably with separation of amnion derived adherent cells. It is well known in the art to select improved postpartum adherent cells from single placenta cells into single cells using enzymes.
  • Attachment culture (eg, P0) of the isolated cell population in a container may comprise cell culture medium, such as Fibroblast Growth Factor-4 (FGF-4) and Culturing in a medium to which heparin is added.
  • FGF-4 in the medium may be added at a concentration of about 10 ng / ml to about 40 ng / ml, or about 20 ng / ml to about 30 mg / ml, for example 25 ng / ml.
  • Heparin in the medium may be added at a concentration of about 0.5 ⁇ g / ml to 2 ⁇ g / ml, or 0.5 ⁇ g / ml to 1.5 ⁇ g / ml, for example 1 ⁇ g / ml.
  • the medium may further include, for example, fetal bovine serum, and antibiotics (eg, penicillin, streptomycin, gentamicin, etc.).
  • antibiotics eg, penicillin, streptomycin, gentamicin, etc.
  • PS-CM medium with 10% fetal bovine serum, 50 ⁇ g / ml gentamicin, 1 ⁇ g / ml heparin, and 25 ng / ml FGF-4 added can be used. Cultivation of the cell population can be performed under hypoxic conditions.
  • hypooxia may refer to a low oxygen partial pressure condition compared to 21% oxygen partial pressure, which is a normal normal oxygen condition.
  • the low oxygen condition may be a state having an oxygen partial pressure of 1 to 15%, 1 to 12%, 1 to 10%, or 1 to 5%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%.
  • the culturing may be performed, for example, for 2 to 7 days, or for 3 to 5 days, and may be treated with a recombinant enzyme without an animal-derived component after the culturing.
  • the term "Animal Component Free Enzyme” is of non-animal origin, which may mean that the enzyme is not purified from an animal source.
  • the enzyme without the animal derived component may be of recombinant origin, for example bacterial, yeast or plant origin.
  • Enzymes of recombinant origin may refer to any enzyme produced by recombinant DNA technology, including the use of microorganisms such as bacteria, viruses, yeast, plants, etc. for their production.
  • the enzyme can be recombinant trypsin without animal derived components, for example recombinant trypsin produced in corn.
  • Recombinant trypsin free of animal-derived components are commercially available and include, for example, TrypLE TM Select (GIBCO Invitrogen), TrypLE TM Express (GIBCO Invitrogen), TrypZean TM (Sigma Aldrich) or Recombinant Trypsin Solution TM (Biological Industries). Can be.
  • the passage number of the passage is not particularly limited and may be appropriately selected according to the number of desired proliferating cells.
  • at least one passage, at least 10 passages may be present, and the clinically necessary number of cumulative proliferating cells may be obtained, for example, by performing 1 to 20 passages, 1 to 6 passages, 1 passage, 3 passages, or 6 passages. You can get it.
  • the improved postpartum adherent cells obtained as described above may be used in a cell culture medium, for example Fibroblast Growth Factor-4, FGF-4) and passaging in a medium to which heparin is added. Fibroblast Growth Factor-4 (FGF-4) and heparin added medium and culture conditions are as described above.
  • FGF-4 Fibroblast Growth Factor-4
  • the treatment of a recombinant enzyme without an animal-derived component may be additionally performed. That is, the purity of adherent cells can be improved by collecting the cells by treating a recombinant enzyme without an animal-derived component before passing the cells to the next step for each passaging step. For example, in a step from P1 to P2, the recombinant enzyme without the animal-derived component may be treated before transplanting the cells for P2.
  • the improved postpartum adherent cells prepared according to the present invention are at least about 20%, 25%, 30%, of CD44, CD73, CD90, and CD105 positive surface markers for adherent cell markers expressed on the cell surface as the cells differentiate and proliferate. Expresses 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99%, hematopoietic cells At least about 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least It may be to express less than 1%.
  • the term “positive” may mean that the label is present in a greater amount, or at a higher concentration, as compared to other cells on which the label is based. That is, a cell is positive for that label because a label is present inside or on the surface of the cell and the label can be used to distinguish the cell from one or more other cell types. It may also mean that the cell has its label in an amount sufficient to give a signal greater than the background value, for example a signal from a cytometry device. For example, if a cell can be detectably labeled with an antibody specific for CD105 and the signal from this antibody is detectably greater than the control (eg background value), then the cell is "CD105 +".
  • the term “negative” means that even when an antibody specific for a specific cell surface label is used, the label cannot be detected in comparison with the background value. For example, if a cell cannot be detectably labeled with an antibody specific for CD45, the cell is "CD45-".
  • the cell population harvesting step uses an enzyme mixture solution to give an improved yield of postpartum adherent cells as compared to without using them. For example, it can be increased by 50 times or more.
  • treatment of a recombinant enzyme without an animal-derived component does not separate dense lump-shaped cells, but does not separate, leaving only homogeneous cells after passage. Can be.
  • culturing under hypoxic conditions shortens the doubling time of the cell, thereby reducing the proliferation rate of the cell, for example, 7 Can be increased more than twice.
  • an improved postpartum adherent cell produced by the method for producing an improved postpartum adherent cell may secrete the proteins described in Table 3 below.
  • the improved postpartum adherent cells are Vascular endothelial growth factor (VEGF), transforming growth factor (TGF) - ⁇ 1, hepatocyte growth factor (Hepatocyte) growth factor (HGF), interleukin-6 (IL-6) or progranulin may be secreted.
  • the enhanced postpartum adherent cells have a pronounced ability to migrate to damaged tissue. Therefore, the improved postpartum adherent cells may be usefully used for the treatment of neurological diseases and other diseases in which the secreted protein may act as a cell therapy.
  • Another aspect includes adding a solution of an enzyme mixture to isolated placental derived tissues to harvest cell populations; Attaching and culturing the harvested cell population to a flask, followed by treatment with an Animal Component Free (ACF) recombinant enzyme to separate enhanced postpartum adherent cells; And passivating the isolated enhanced postnatal adherent cells in a medium comprising fibroblast growth factor-4 (FGF-4) and heparin at low oxygen conditions compared to 21% normal oxygen conditions.
  • ACF Animal Component Free
  • the method for producing the enhanced postpartum adherent cells is as described above.
  • increased production of postpartum adherent cells can be achieved by increasing the yield of adherent cells as compared to not using the enzyme mixture solution, and by increasing the proliferation rate of adherent cells as compared to the normal oxygen conditions. Or increase the purity of adherent cells as compared to not using the recombinant enzyme without the animal-derived component.
  • the increase in manufacturing efficiency may include not only an increase in the efficiency of the manufacturing method itself, but also an increase in useful properties of the prepared adherent cells, for example, as described above in adherent cells prepared according to one embodiment. Increased production of proteins useful for treating diseases or increased ability to migrate to damaged tissue.
  • Another aspect includes obtaining placental derived tissue from an isolated placenta; Collecting a cell population by adding an enzyme mixture solution to the placental tissue; Attaching and culturing the harvested cell population to a container, and then treating the animal component free (ACF) recombinant enzyme to separate enhanced postpartum adherent cells; And passivating the isolated enhanced postnatal adherent cells in a medium comprising fibroblast growth factor-4 (FGF-4) and heparin at low oxygen conditions compared to 21% normal oxygen conditions.
  • FGF-4 fibroblast growth factor-4
  • the method is as described above.
  • the improved postpartum adherent cells prepared by the method may have at least about 20%, 25%, 30%, 35%, 40% CD44, CD73, CD90, and CD105 positive surface markers for adherent cell markers expressed on the cell surface.
  • the improved postpartum adherent cells produced by the method may be to secrete the proteins listed in Table 3 below, and may be increased ability to migrate to damaged tissue.
  • Another aspect provides a cell therapeutic or pharmaceutical composition comprising said enhanced postpartum adherent cells (ePACs).
  • ePACs enhanced postpartum adherent cells
  • Another aspect provides a method of preventing or treating a disease comprising administering the improved postpartum adherent cells (ePACs) or a cell therapy or pharmaceutical composition comprising the same to an individual in need thereof.
  • ePACs improved postpartum adherent cells
  • Another aspect provides the use of said enhanced postpartum adherent cells (ePACs) for use in the manufacture of a medicament for the prevention or treatment of a disease.
  • ePACs enhanced postpartum adherent cells
  • Enhanced postpartum adherent cells secrete proteins advantageous for treating a disease as described above, and because the ability to move to damaged tissues is remarkable, cell therapeutics or pharmaceuticals for the prevention or treatment of various diseases It can be usefully used in the composition.
  • Examples of such diseases may include neurological diseases, liver diseases or metabolic diseases.
  • Examples of such nervous system diseases include Alzheimer's disease, chronic or acute stroke, cerebral infarction, brain tumor, cerebral edema, cerebral ischemia, multiple sclerosis, Frontotemporal dementia, progressive supranuclear palsy, cortex Corticobasal degeneration, Pick's disease, or dementia pugilistica (DP).
  • Examples of such metabolic diseases may include obesity, type 1 or type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis or nonalcoholic fatty liver.
  • the dosage of the cell therapy or pharmaceutical composition may be from 1.0 X 10 3 to 1.0 X 10 10 cells / kg (body weight) or individual, or from 1.0 X 10 7 to 1.0 X 10 8 cells / based on adherent cells. kg or body weight.
  • the dosage may be variously prescribed by such factors as the formulation method, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient. These factors can be taken into account to properly adjust the dosage.
  • the number of administrations may be one or two or more times within the range of clinically acceptable side effects, and may be administered to one or two or more sites of administration.
  • the amount converted into the amount can be administered.
  • the target animal for the treatment according to one embodiment include humans and mammals for other purposes, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, and the like. Included.
  • the cell therapeutic agent or pharmaceutical composition may include the adherent cells and a pharmaceutically acceptable carrier and / or additive as an active ingredient.
  • a pharmaceutically acceptable carrier examples include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do.
  • organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof.
  • the cell therapy or pharmaceutical composition according to one embodiment is formulated in a formulation suitable for injection, the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in solution.
  • the cell therapeutic agent or pharmaceutical composition according to one embodiment may be prepared according to the administration method or dosage form thereof, and as necessary, suspending agents, dissolution aids, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, A passivating agent, a buffer, a reducing agent, an antioxidant, etc. can be included suitably.
  • suspending agents, dissolution aids, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, A passivating agent, a buffer, a reducing agent, an antioxidant, etc. can be included suitably.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
  • the cell therapeutic agent or pharmaceutical composition according to one embodiment is formulated with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by one of ordinary skill in the art. It may be prepared in unit dose form or incorporated into a multi-dose container. The formulations can then be in the form of solutions, suspensions or emulsions in oil or aqueous media or in the form of powders, granules, tablets or capsules.
  • an improved postpartum adherent cell isolation and culture method it is possible to increase the purity, yield and proliferation rate of adherent cells from placental tissue, to secrete proteins effective for neurological diseases, and to move to damaged tissues.
  • This improved adherent cell can be prepared.
  • the adherent cells of the present invention possess high differentiation capacity and proliferative capacity and can maintain suitable properties as cell therapeutics.
  • Figures 1a and 1b is a view showing the homogeneity and surface antigen characteristics of the method for producing adherent cells according to one embodiment.
  • Figure 2a and 2b is a view showing the cell yield according to the components of the enzyme reaction solution of the method for producing an adherent cell according to one embodiment.
  • 3A to 3C are diagrams comparing and analyzing characteristics according to culture conditions (hypoxia and normal oxygen conditions) of the method for preparing an adherent cell according to one embodiment.
  • Figures 4a and 4b is a view showing the morphological characteristics and genetic safety of the adherent cells prepared by the method for producing adherent cells according to one embodiment.
  • Figures 5a to 5e is a view showing the result of quantitative analysis of secretion proteins of adherent cells prepared by the adherent cell production method according to one embodiment according to passage culture.
  • reaction enzyme solution 2 ml FBS was added at a ratio of 1:10, and the reaction solution was centrifuged at 1,500 rpm for 3 minutes, and then the supernatant was transferred to a new tube. The procedure was repeated twice for the remaining tissue.
  • the lysed tissues were isolated from amnion cells using a 100 ⁇ m cell sieve.
  • the components of the enzyme reaction solution (Enzmye mixture) and the components of the PS-CM medium used are shown in Tables 1 and 2, respectively.
  • 1 is a view showing the homogeneity and surface antigen characteristics of the method for producing an adherent cell according to one embodiment.
  • adherent cells were separated from the amnion tissue of the placenta using the enzyme reaction solution of Table 1 and three enzyme reaction solutions of the comparative control, respectively, and the cells separated from each enzyme reaction solution were compared to compare their yields.
  • T-flask inoculation was incubated in 37 °C, low oxygen culture conditions (CO 2 5%, O 2 3%). Every 3 to 4 days, PS-CM medium was replaced to remove cells that did not adhere to the bottom of the flask, and then cultured in the cell group separated by the enzyme reaction solution of Table 1 until the cells formed a colony, and then cells from each T-flask.
  • Figure 2 is a view showing the cell yield according to the components of the enzyme reaction solution of the method for producing an adherent cell according to one embodiment.
  • the enzyme reaction solution according to Table 1 was significantly higher yield of amnion derived adherent cells, especially collagenase compared to collagenase exclusion group, trypsin exclusion group, and dispase exclusion group.
  • the cell harvesting efficiency was increased by about 50 times or more with the exclusion group and about 5 to 6 times or more with the trypsin exclusion group.
  • adherent cells cultured in hypoxic conditions were compared with adherent cells cultured in normal oxygen conditions.
  • the adherent cells were isolated and cultured under normal oxygen conditions in the same manner.
  • the same number of cells were inoculated into 6 well plates to determine the plate bottom area. The cells were collected when occupied 70 to 80% and the number of cells was measured. A total of three measurements were performed. The total cell number was mixed with 10 ⁇ l of the cell suspension and 10 ⁇ l of trypan blue, and then 10 ⁇ l was counted using a hemocytometer.
  • Doubling time was calculated by using the total number of cells and the time when the number of cells reached twice the number.
  • in order to analyze the ability of the cells to move to the damaged tissue (transwell) was used to confirm the cell migration capacity. After inoculating each cell with the same number on the top of the transwell, chemokine was added to the lower cell culture. After culturing for several days, the number of cells moved through the transwell was counted to confirm cell migration. The results are shown in FIG. 3.
  • Figure 3 is a comparison of the characteristics according to the culture conditions (hypoxia and normal oxygen conditions) of the method for producing an adherent cell according to one embodiment.
  • the cell morphology was observed at 40 ⁇ magnification using an inverted microscope (Eclipse TS100 (Nikon)).
  • the cells were confirmed to exhibit the specific morphology of spindle-shaped fibroblasts with irregular processes, and karyotyping was analyzed by G-banding method (Gosden JR (1994) Chromosome analysis protocols.In: Walker JM (ed) ., Methods in Molecular Biology, vol. 29. Totawa: Humana Press., Sumner AT (1990) Chromosome Banding. London: Unwin Hyman.
  • chromosomal aberration of adherent cells at P1, P3, and P6 was analyzed using SNPs (Single Nucleotide polymorphisms) method.
  • SNPs Single Nucleotide polymorphisms
  • DNA was extracted from cells of each passage using a Promega DNA Extraction Kit and used as a sample.
  • An Illumina HumanOmni1-Quad Chip was used and measured using an iSCAN® scanner.
  • 400 ng of each DNA sample is amplified by whole genome amplification, randomly fragmented by chemical method, purified by 2-propanol precipitation, and the chip is buffered before loading the DNA sample. DNA samples were added to the chips pretreated with the solution.
  • Figure 4 is a view showing the morphological characteristics and genetic safety of adherent cells prepared by the method for producing adherent cells according to one embodiment.
  • cells cultured up to P14 is well maintained morphologically, it can be confirmed that it has a fibroblast-specific morphology with irregular projections, and also there is no genetic variation.
  • adherent cells cultured up to P1, P3, and P6 can be confirmed that there is no abnormality in the chromosome.
  • Example 2 improved postpartum adherent cells Secretory protein Analytical Profiling and Quantitative Analysis
  • Secretory protein profiling was performed to analyze the secreted proteins of adherent cells prepared in Example 1-1.
  • the cultured adherent cells were secreted in MEM alpha GlutaMAX (Invitrogen), a medium without serum, and the secreted protein was concentrated to 1 mg / ml.
  • the concentrate was used to analyze proteins secreted from adherent cells through a human antibody array (Raybio) capable of analyzing 504 secreted proteins.
  • Some specific proteins of the secreted proteins are effective in neurological diseases, and the proteins VEGF, TGF- ⁇ 1, Progranulin, HGF and IL-6, which are useful for regenerating damaged tissues, were secreted. Quantitative analysis.
  • TGF- ⁇ 1 includes the pretreatment of the sample. All were measured using a microplate reader Epoch (BioTek Inc.) at 450 nm and analyzed using Gen5 (2.00) software and the results are shown in FIG. 5.
  • 5 is a view showing the results of quantitative analysis of secretory proteins of adherent cells prepared by the adherent cell preparation method according to one embodiment according to passage culture.
  • VEGF and IL-6 showed an increasing pattern as the passage progressed, and it was confirmed that TGF- ⁇ 1 and progranulin were secreted at 1,000 pg / ml or more.
  • the adherent cells prepared according to one embodiment can be usefully used for cell therapy by secreting them into proteins specific for neurological diseases.

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Abstract

La présente invention concerne des cellules adhérentes postnatales et un procédé de préparation de celles-ci. Le procédé de préparation de cellules adhérentes postnatales améliorées peut augmenter le rendement et la vitesse de prolifération de cellules adhérentes issues de tissus placentaires ; et préparer des cellules adhérentes, qui sécrètent des protéines efficaces pour des maladies neurologiques et qui présentent une capacité améliorée de déplacement vers les tissus endommagés.
PCT/KR2016/005636 2015-05-28 2016-05-27 Cellules adhérentes postnatales améliorées et procédé de préparation associé WO2016190704A1 (fr)

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US15/577,633 US10479978B2 (en) 2015-05-28 2016-05-27 Postnatal adherent cells and preparation method therefor

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KR20150075374 2015-05-28
KR10-2015-0075374 2015-05-28
KR1020160065593A KR20160140492A (ko) 2015-05-28 2016-05-27 향상된 산후 부착형 세포 및 그의 제조 방법
KR10-2016-0065593 2016-05-27

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CN115322964A (zh) * 2022-08-18 2022-11-11 宁波希诺赛生物科技有限公司 一种3d培养羊膜间充质干细胞种子库构建方法
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