WO2014102730A1 - Chambre de perfusion de culture tridimensionnelle pour le génie tissulaire - Google Patents

Chambre de perfusion de culture tridimensionnelle pour le génie tissulaire Download PDF

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WO2014102730A1
WO2014102730A1 PCT/IB2013/061341 IB2013061341W WO2014102730A1 WO 2014102730 A1 WO2014102730 A1 WO 2014102730A1 IB 2013061341 W IB2013061341 W IB 2013061341W WO 2014102730 A1 WO2014102730 A1 WO 2014102730A1
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perfusion
cells
culture
tissue
bioreactor
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Portuguese (pt)
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Marivalda de Magalhães PEREIRA
Alexandra Rodrigues Pereira da SILVA
Alfredo Miranda de GÓES
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Universidade Federal De Minas Gerais - Ufmg
Fundação De Amparo À Pesquisa Do Estado De Minas Gerais- Fapemig
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • the present technology describes a tissue culture perfusion chamber whose fabrication material may involve different types of polymers having good thermal and electrical properties, moderate strength, easy machining and autoclaving, an essential factor when the proposal is to work. with biological material.
  • the camera can have different formats, provided that respecting the conditions of cultivation of the fabric of interest.
  • the developed perfusion chamber makes it possible to perform a three-dimensional culture of seeded cells in a matrix which, when properly adapted to the perfusion chamber, favors the flow of flow through this scaffold, ensuring that the medium can reach most if not all. thus favoring cell viability and proliferation.
  • Tissue engineering was defined as a research field only in the early 1980s and today progress in this field is related to the development of various study model approaches. Tissue engineering supports its scientific base in many areas of knowledge, such as cell biology, biochemistry, molecular biology, materials science, chemical engineering and bioengineering, which allow the rational application of engineering principles in living systems.
  • the scientific challenge of tissue engineering involves understanding cellular mechanisms and fabricating materials to provide matrices and models that guide tissue regeneration (VACANTI, J .; VACANTI, C.
  • VACANTI The History and Scope of Tissue Engineering. Principles of Tissue Engineering , 3rd ed., Elsevier, Inc, Chapter 1, pp. 3-6,).
  • Living stem cells are required for the manufacture of tissue substitutes. These cells create or recreate functional structures using preprogrammed information and signaling in their genetic code.
  • the acquisition of cells for the production of organic structures is a major challenge, because for the manufacture of large-scale structures, there is a need to obtain large numbers of cells that are immunologically compatible with the recipient individual (VACANTI, J.; VACANTI, C.
  • VACANTI J.
  • VACANTI C.
  • Bone marrow contains mesenchymal stem cells (MSC), but harvesting is an invasive procedure.
  • Adipose mesenchymal stem cells are able to differentiate into chondrogenic, adipogenic, myogenic and osteogenic lineages, suggesting their applicability in tissue reconstitution (ZUK, PA; ZHU, M .; MIZUNO, H .; HUANG, J .; FUTRELL, JW; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 211-228, 2001.).
  • Bioreactors are the main tools to mimic cellular microenvironments and provide cellular constructs with physiologically relevant stimuli that direct the conversion of a "set of cells" into a given tissue (FRESHNEY, RI; OBRADOVIC, B .; GRAYSON, W .; CANNIZZARO, C; VUNJAK-NOVAKOVIC, G. Principles of Tissue Culture and Bioreactor Design. Principies of Tissue Engineering, 3rd ed., Elsevier, Inc, Chapter 12, p. 155-183, 2007).
  • the bioreactor-induced hydrodynamic shear is believed to simulate interstitial flow in the canalicular spaces during natural bone formation associated with compression loading.
  • the resulting mechanical forces are an integral part of bone physiology that regulates homeostasis, bone remodeling and repair, however few studies apply mechanical deformation to explore their role in promoting cell growth and tissue organization (ALLORI, A. C; SAILON, AM; PAN).
  • JR WARREN SM Biological Basis of Bone Formation, Remodeling, and Repair — Part III: Biomechanical Forces, Tissue Engineering: Part B, v. 14, no. 3, pp. 285-293, 2008).
  • perfusion chambers in a bioreactor system are intended to increase the supply of nutritive material to cells and remove metabolites generated by cell activity, thereby enabling a favorable environment for cell growth (Dick, D. An Easily Made Tissue Culture Perfusion Chamber, Quarterly Journal of Microscopical Science (363, 1995). Since its invention in the mid-1907, as new cell and tissue culture techniques have emerged, new models of perfusion chambers have been developed (Wolf, D; Sluder, G. Methods in Celi Biology, v. 56, p. 256 , 1998). Currently, there is still no consensus in the literature on a standard perfusion chamber and bioreactor model for tissue engineering for cell engineering and several laboratories around the world are looking for this.
  • the present work has as main objective the construction of a perfusion chamber suitable for tissue culture, basing its efficiency on an analysis of the cellular differentiation of human adipose tissue stem cells, sown in a glass foam matrix. bioactive and grown in a bioreactor system for the production of a construct to be applied in tissue engineering.
  • bioactive glass foams produced by the sol-gel process (Hiratsuka, R. et al.) The process solgllma bibliographic review, p. 171 - 179, 1994).
  • COELHO MB Development of methodology for the production of porous three-dimensional bioactive glass structures for application in tissue engineering. Belo Horizonte: UFMG School of Engineering, 2003. 145p.
  • the development of the present technology proved to be very efficient for the cultivation of stem cells derived from human adipose tissue which, when sown in bioactive glass, were able to proliferate and differentiate into the phenotype of interest, producing typical osteogenic cell proteins and expressing genes. characteristic of osteoblasts. This efficiency can be expanded to other tissues as long as conditions, flow rate, culture matrix and perfusion chamber dimensions are adapted.
  • Figure 1 Illustration of bioreactor organization scheme: Greenhouse at 37 Q C (1), Biomaterial (2), Infusion Chamber (3), Culture Medium + O2 (4), Peristaltic Pump (5), Connection Tubes Inside the CO 2 Greenhouse (6).
  • Figure 2 Shows a non-limiting representation of the perfusion chamber.
  • Figure 3 Newly seeded hASC bioactive glass foam.
  • FIG. 4 Scanning electron microscopy (SEM) images at 21 days of cultivation in the bioreactor showing: (A) the surface of the bioactive glass sample with several attached cell clusters (indicated by arrows); (B) a pore of the sample with several adhered cells, (C) larger pore enlargement showing better cells and material roughness, and (D) greater increase in material-adhered hASC and secretion granules in the cell plasma membrane.
  • SEM scanning electron microscopy
  • Figure 5 Representative graph of the viability and proliferation of hASC grown in normal (static) medium and in bioactive (dynamic) glass matrices containing leibovitz (LEI) or leibovitz medium plus osteogenic factors (LEI O in the caption: static O and dynamic O). All times between static and dynamic and between static O and dynamic O are p ⁇ 0.001).
  • Figure 6 Representative graph of alkaline phosphatase activity of hASC grown in bioactive glass matrices containing leibovitz medium (LEI) or medium leibovitz plus osteogenic factors (LAW O in the caption: static O and dynamic O).
  • LAI leibovitz medium
  • LAW O medium leibovitz plus osteogenic factors
  • Figure 7 Immunofluorescence analysis for (A) osteocalcin, (B) osteopontin and (C) hASC type I collagen seeded in bioactive glass matrix and cultured in basal culture medium in perfusion bioreactor after 21 days. Cell nuclei have blue marking and specific proteins green.
  • Figure 8 Immunofluorescence analysis for hASC osteocalcin protein seeded in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
  • Figure 9 Immunofluorescence analysis for hASC osteopontin protein sown in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
  • Figure 10 Immunofluorescence analysis for hASC type I collagen protein sown in bioactive glass matrix and cultured in osteogenic culture medium in perfusion bioreactor after (A) 7, (B) 14 and (C) 21 days.
  • Figure 11 Detection of osteocalcin, osteopontin and alkaline phosphatase at 7, 14 and 21 days in hASC cultured on the bioactive glass matrix, LEI O medium and perfusion biorector. Human osteosarcoma (SOS) cells as positive control and C- is negative control.
  • SOS Human osteosarcoma
  • the present technology consists of a tissue culture perfusion chamber (Figure 2), the fabric of which may involve different types of polymers which have good thermal and electrical properties, moderate strength, easy machining and can be autoclaved, an essential factor. when the proposal is to work with biological material.
  • a tissue culture perfusion chamber (Figure 2), the fabric of which may involve different types of polymers which have good thermal and electrical properties, moderate strength, easy machining and can be autoclaved, an essential factor.
  • polypropylene is used, a low density resin that offers a good balance of thermal and electrical properties, as well as moderate strength, is nontoxic, easily machined and allows autoclaving.
  • the camera can have different formats and dimensions, as long as it respects the conditions of interest.
  • the matrix of choice for the cultivation of a given tissue must be correctly adapted to the perfusion chamber to favor the flow of the flow through the matrix, ensuring that the medium can reach most if not all cells, thus favoring the viability and cell proliferation.
  • the perfusion chamber was designed with one inlet and one outlet (7 and 9, respectively) to allow perfusion of the culture medium throughout its interior, where the construct (biomaterial and seeded cells) must be adequately adapted.
  • the matrix material (2) as well as the chamber shape vary according to the cell type of the tissue chosen for cultivation.
  • the perfusion chamber ( Figure 2) developed can be maintained in different environments, from a B.O.D (Biochemical Oxygen Demand) greenhouse to a water bath, provided the necessary conditions for cultivation are respected.
  • the perfusion chamber proposed to compose the bioreactor system (3) was able to efficiently promote bone tissue culture.
  • the developed perfusion bioreactor (1) is a closed, passive sterilization system where the cell culture medium is constantly pumped at a constant flow rate through a peristaltic pump (5) and through connected hoses (6) to one end of the infusion chamber.
  • the pumped medium passes through the construct and returns through the connecting hoses, connected at the other end of the chambers, to the culture medium reservoir as shown in Figure 1.
  • the tubes chosen to connect the bioreactor system are calibrated silicone peristaltic tubes with 2.6 mm internal diameter. These tubes were chosen because they are autoclavable, translucent and have biocompatibility and high gas permeability.
  • the reservoir for the culture media is screw-capped glass bottles. In the bottle caps, three holes were drilled, two of them for fitting the connection hoses - one for entering and one for leaving the culture medium - and one for fitting a breather filter to avoid the formation of vacuum inside the bottles during the tests and possibility of being another way of gas exchange.
  • the peristaltic pump of choice produces smooth, pulse-free flow, has 8 channels, allowing up to 8 perfusion chambers to work simultaneously, fed by a constant flow of culture medium from 0.05mL / min to 40mL / min per channel.
  • the bioreactor culture medium was partially changed every seven days during cell culture, and only half of the medium was removed in order not to eliminate the new components produced by cells, such as growth factors, hormones and enzymes, which are important. in the regulation of the cellular mechanisms themselves (GOLDSTEIN, AS; JUAREZ, TM; HELMKE, CD; GUSTIN, M. C; Mikos, AGE). Convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials, v. 22p. 1279 - 1288, 2001; MARTIN, Y; VERMETTE, P. Bioreactors for Tissue Mass Culture: Design, Characterization, and Recent Advances. Biomaterials, v. 26, pp. 7481-7503, 2005.).
  • the physiological conditions of bioreactor temperature and pH were controlled.
  • the temperature of 37 Q C was assured by maintaining the hose and reservoirs in the culture medium bath and monitored daily with the aid of a thermometer.
  • Already the pH was monitored with each change of culture medium with the aid of a pH meter.
  • the method chosen for sterilization of bioreactor accessories was autoclaving.
  • Adipose tissue stem cells were used for differentiation into bone tissue.
  • the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8).
  • the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8).
  • the chamber was made of polypropylene and in a cylindrical shape, obeying the following internal dimensions: 7mm in diameter and 10mm in length (8).
  • conduction tubes are connected which connect the chamber to the culture medium and a propellant pump (7 and 9, respectively).
  • the hASCs were grown on bioactive glass foam matrix, with dimensions adapted to the bioreactor dimensions, and were placed in individual perfusion chambers, each connected to a reservoir. containing Leibovitz medium plus osteogenic factors and kept in a water bath at 37 ° C for 7.14 and 21 days.
  • bone tissue we analyzed: cell viability and proliferation, alkaline phosphatase enzyme activity, osteogenic cell markers, as well as morphology and gene expression.
  • cell viability and proliferation we analyzed: cell viability and proliferation, alkaline phosphatase enzyme activity, osteogenic cell markers, as well as morphology and gene expression.
  • the biomaterial chosen for the cultivation of hASC was processed, in this case, the bioactive glass foam, and its characterization so that its chemical and structural characteristics could be verified.
  • the structure of the bioactive glass foams was preliminarily evaluated by scanning electron microscopy (SEM) and it was observed that they had high porosity, with spherical pores, average pore sizes ranging from 100 to 500pm and an interconnected pore network. .
  • the analysis performed in various areas of bioactive glass foams detected the presence of the chemical elements silicon (Si), calcium (Ca), phosphorus (P) and gold (Au), the latter being due to the matrix metallization process.
  • the X-ray diffractogram analysis of the bioactive glass foam sample presents a characteristically amorphous material.
  • the perfusion bioreactor system was adapted for use in a water bath in order to maintain the temperature of 37 Q C, ideal for cell culture ..
  • To ensure the maintenance of the pH of the cell culture was Leibovitz tested Independent CO 2, which is able to maintain pH stability in the long term under atmospheric CO 2.
  • the hASCs isolated from the stromal vascular fraction resulting from the liposuction processing showed satisfactory results, presenting cells with fibroblast-like shape and adhering to the plastic surface, corroborating the data of Zuk (ZUK, PA; ZHU, M .; MIZUNO, H. ; HUANG, J .; FUTRELL, JW; KATZ, AJ; BENHAIM, P.; LORENZ, HP; HEDRICK, MH Multilineage Celis from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Engineering, 7, 21 1 -228, 2001).
  • Example 2 Since the samples used in the static tests (Example 2) were 3mm long and the samples grown in the perfusion bioreactor were 10mm, all samples were cut into three parts and subjected to subsequent biological analysis to allow comparison of the static culture results. and dynamic. After 7, 14 and 21 days, colonized matrices were prepared for SEM analysis to confirm the presence and adhesion of hASC to the surface and interior of colonized foams cultured in the perfusion bioreactor ( Figure 4).
  • Formazan crystals are solubilized and the optical density can be determined by spectrophotometer at 595 nm.
  • the number of viable cells is directly proportional to the amount of formazan crystals produced.
  • HASC-colonized bioactive glass foams cultured in the LEI and LEI O perfusion bioreactor were evaluated after each culture interval for hASC alkaline phosphatase activity by the BCIP / NBT assay ( Figure 6) based on chromogenic reaction resulting from the cleavage of a phosphate group BCIP (5-bromo 4-chloro 3-indolylphosphate p-toluidine) by the alkaline phosphatase produced by the cell.
  • hASC osteogenic lineage cells when cultured in bioactive glass matrices and perfusion bioreactor was evaluated by the expression of specific proteins produced by osteoblasts using the immunofluorescence technique.
  • cells were washed with PBS and fixed in 4% p-formaldehyde for 15 minutes at room temperature.
  • Cells were washed with PBS and plasma membrane permeabilization was performed using 0.1% Triton-100x in PBS for 10 minutes. After permeabilization, the cells were washed again with PBS. The reaction was then blocked by incubating the cells with 1% BSA (bovine serum albumin) and 5% goat serum in PBS for 1 hour at room temperature.
  • BSA bovine serum albumin
  • cells were incubated with primary antibody diluted in 1% BSA solution in PBS for 2 hours at room temperature and in a humid chamber. After incubation with primary antibody, the cells were again washed with PBS and then incubated with their secondary antibodies diluted in 1% BSA 1% solution in PBS for 1 hour in a humid chamber at no light and at room temperature. Negative controls were made using only the respective secondary antibody. Subsequently, cells were washed with PBS and incubated with the Hoechst probe (1 pg / ml) (Molecular Probes) for 30 minutes for nucleus labeling. Further washes were made with PBS and the coverslips were mounted with Hydromount on slides.
  • Hoechst probe (1 pg / ml) (Molecular Probes) for 30 minutes for nucleus labeling.
  • the slides were visualized and analyzed through the Confocal Microscope (Zeiss LSM 510 Meta) using the Carl Zeiss Laser Scanning Microscope LSM 510 ⁇ software.
  • the assay used to verify the expression of osteogenesis-related genes was the Polymerase Chain Reaction (PCR).
  • the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered as a control for reaction amplification.
  • the hASCs sown in the bioactive glass were grown in the bioreactor in LEI O medium for 7, 14 and 21 days and, after each period, RNA extraction was performed.
  • RNA extraction from hASC was attempted by applying 1 ml Trizol (Gibco) on the bioactive glass matrices, incubating for 5 minutes at room temperature. The formed solution was processed following the RNA extraction steps usually used for cells grown in culture bottles, but when the absorbance reading at 260 / 280nm was taken, the results were close to zero.
  • aHASC-seeded bioactive glass samples taken from the bioreactor were cut into three parts and each into four parts and trypsinized for 5 minutes in a CO 2 oven so that the cells could be removed from inside the biomaterial. During trypsinization, the samples were gently shaken every 2 minutes. After trypsinization, the biomaterial was washed three times with LEI medium and the total solution was centrifuged for 5 minutes at 252 g. The pellet formed was resuspended in 1 ml Trizol and incubated for 5 minutes for cell lysis and homogenization at room temperature and for complete dissociation of nucleoprotein complexes.
  • the formed solution was transferred to a microtube 1, 5 mL and centrifuged for 15 minutes at 4 Q C and 12,000 g.
  • the upper phase was collected and transferred to a new microtube 1, 5mL which was added 0.2 ml of chloroform, followed by homogenization, two minutes incubation at room temperature and centrifugation at 12,000 g for 15 minutes at 4 Q C to three-phase separation where the superficial colorless phase contained the RNA.
  • the colorless phase was transferred to a new microtube and the RNA It was precipitated with 0.5 ml of isopropyl alcohol for 10 minutes at room temperature.
  • RNA was solubilized in DNAse and RNAse free distilled water and immediately stored at -80 Q C. RNA concentration was determined by reading the absorbance at 260 / 280nm in Nanodrop.
  • CDNAs were synthesized from total RNAs using the RevertAid TM H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's recommendations. Two micrograms of total RNA from each sample were incubated with 0.5 ug oligo (dT) 18 at 65 Q C for 5 minutes and then the samples were incubated on ice. Immediately after it was added to the samples: 5X reaction buffer, 20 units Ribolock TM Ribonuclease Inhibitor, 10mM dNTP mix and incubated at 37 Q C for 5 minutes. Then it was added 200 units of enzyme RevertAid TM H Minus M-MuLV RT (Fermentas) and the samples were incubated for 60 minutes at 42 Q C. The reaction was stopped by heating at 70 Q C for 10 minutes.
  • RevertAid TM H Minus First Strand cDNA Synthesis Kit Fermentas
  • the bioactive glass has had a osteoinductive action represented by the increased production of the enzyme in the period 14 days and decrease of this production in the 21 Q day, suggesting a probable compromise with the osteogenic phenotype.
  • the flux had a synergistic action to that of bioactive glass, which demonstrated an increase of alkaline phosphatase synthesis in the bioreactor constructs.
  • the immunofluorescence assay was performed to verify whether hASCs sown in bioactive glass foams and cultured in the perfusion bioreactor began to express osteogenic cell markers.
  • the specific antibodies used were osteocalcin, osteopontin and type I collagen and initially the test was performed on cells cultured in LEI medium after 21 days of culture. It can be observed in the confocal microscopy images that the hASC were labeled for the three proteins tested ( Figure 7). This result corroborates the results of MTT and alkaline phosphatase activity, where, from 14 Q day, there was a decrease in cell proliferation and an increase in the production of alkaline phosphatase enzyme, suggesting a compromise of hASC with osteogenic phenotype. .
  • confocal microscopy images also show a decrease in hASC length, also suggesting a change in morphology related to change in phenotype ( Figure 8).
  • osteogenic differentiation of HASC when cultured on bioactive glass matrix and means ACT in the perfusion bioreactor can be suggested that from 14 Q day of cultivation, the HASC seem to have committed to the osteogenic phenotype.
  • the evaluation of osteocalcin, osteopontin and collagen type I differentiation markers was performed in hASC grown on bioactive glass in LEI O medium for 7, 14 and 21 days in the perfusion bioreactor.
  • the confocal microscopy images show an increase in fluorescence labeling of from 7 to 21 Q day for three tested proteins: osteocalcin (Figure 8), osteopontin (Figure 9) and collagen type I ( Figure 10);
  • the cells move from the typical fusiform shape of a stem cell to the cuboidal shape, characteristic of osteoblastic cells.
  • hASC sown in the three-dimensional bioactive glass matrix and cultured in LEI O medium in the bioreactor showed a compromise with the osteogenic phenotype.

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Abstract

La présente technologie décrit une chambre de perfusion pour culture de tissus, dont le matériau de fabrication peut comprendre différents types de polymères possédant de bonnes propriétés thermiques et électriques, une résistance modérée, ce matériau étant facile à usiner et pouvant être autoclavé, facteur essentiel lorsqu'il est proposé de travailler avec une matière biologique. Dans son aspect physique, la chambre peut présenter différentes formes, les conditions de culture du tissu d'intérêt étant toujours respectées. La chambre de perfusion mise au point permet de réaliser une culture tridimensionnelle de cellules inoculées dans une matrice qui, lorsqu'elle est correctement adaptée à la chambre de perfusion, favorise l'écoulement à travers cet échafaudage, garantissant que le milieu puisse parvenir la majorité, voire à la totalité, des cellules, la viabilité et la prolifération cellulaire étant ainsi favorisées.
PCT/IB2013/061341 2012-12-28 2013-12-26 Chambre de perfusion de culture tridimensionnelle pour le génie tissulaire WO2014102730A1 (fr)

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Cited By (3)

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WO2019051486A1 (fr) * 2017-09-11 2019-03-14 3D Biotek, Llc Bioréacteur à grande échelle
WO2022068029A1 (fr) * 2020-09-29 2022-04-07 中国肉类食品综合研究中心 Appareil de culture spécial pour tissu biologique 3d, et procédé de préparation de viande cultivée en forme de bloc
US11566215B2 (en) 2016-08-27 2023-01-31 3D Biotek Llc Bioreactor with scaffolds

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US11566215B2 (en) 2016-08-27 2023-01-31 3D Biotek Llc Bioreactor with scaffolds
US11926810B2 (en) 2016-08-27 2024-03-12 3D Biotek, Llc Bioreactor with scaffolds
WO2019051486A1 (fr) * 2017-09-11 2019-03-14 3D Biotek, Llc Bioréacteur à grande échelle
WO2022068029A1 (fr) * 2020-09-29 2022-04-07 中国肉类食品综合研究中心 Appareil de culture spécial pour tissu biologique 3d, et procédé de préparation de viande cultivée en forme de bloc

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