| 摘要 |
<p>#CMT# #/CMT# Dynamic breeding, reproduction and/or differentiation of stem cells or primary cells of human and animal origin, comprises: adhering the stem cells or primary cells of human and animal origin on cell carriers or implant surface, culturing by dynamic supply of nutrient medium, carrying out the cultivation, propagation or differentiation in an overlay-atmosphere; dynamically cultivating the adherent cells on cell carriers or implant surface; and simultaneously subjecting to constant and/or fluctuating pressure load. #CMT# : #/CMT# Dynamic breeding, reproduction and/or differentiation of stem cells or primary cells of human and animal origin, comprises: adhering the stem cells or primary cells of human and animal origin on cell carriers or implant surface, culturing by dynamic supply of nutrient medium, carrying out the cultivation, propagation or differentiation in an overlay-atmosphere, where a rotating bed (6) driven by a magnetic actuator is designed as the cell carriers or implant surface in a reactor housing (1); dynamically cultivating the adherent cells on cell carriers or implant surface; and simultaneously subjecting to constant and/or fluctuating pressure load. The cell carrier or implant surface is rotationally moved through the culture medium in the reactor housing and through the overlay atmosphere in the head-space of the reactor housing alternately at a constant rotation speed of the rotating bed of 0.1-20 revolutions per minute, preferably 0.5-2 revolutions per minute. An independent claim is also included for a pressure-rotating bed bioreactor for dynamic breeding, reproduction and/or differentiation of stem cells or primary cells of human and animal origin, comprising a pressure-resistant reactor housing made of borosilicate glass with pressure-resistant lids, which are detachably arranged on the sides, tightly fastened to the reactor housing and are made of stainless steel, where a storehouse is provided in the two lids (2, 3) for accommodating a shaft or a receiving device (4). The rotating bed comprising the cell carriers or implant surface is attached to the shaft or receiving device. The rotating bed is used as a package of cell carriers or implant surface arranged over one another, with defined distance from each other parallel to the shaft or receiving device of the rotating bed through the rectangular slabs, which are supported by lateral receivers different respectively to the diameter of the reactor housing adjusted width or as a package of cell carrier or implant surface with round discs having defined spaces. The rotating bed is arranged with adjusted diameter of the reactor housing, which is arranged perpendicular to the shaft or receiving device on the shaft or receiving device or other conventional cell carriers or implant surface, which are used as rotating beds. The rotating beds are driven by a permanent magnet (7) arranged on the shaft or receiving device, or rotated by magnetic actuator outside the reactor housing in rotation. The lids are provided with ports for the supply and discharge of culture- and nutrient medium. The ports for the supply and discharge of overlay atmosphere are provided in the inlets and outlets for the culture- and nutrient medium. The pressure control valves are arranged in proportional technology for inlet and outlet of overlay atmosphere. #CMT#USE : #/CMT# The method is useful for dynamic breeding, reproduction and/or differentiation of stem cells or primary cells of human and animal origin. #CMT#ADVANTAGE : #/CMT# The method provides conditions equivalent to the in-vivo conditions and mechanically stable implants. #CMT#DESCRIPTION OF DRAWINGS : #/CMT# The figure shows a schematic representation of the pressure-rotating bed bioreactor with round discs as cell culture carrier perpendicular to the shaft of the rotating bed. 1 : Reactor housing 2, 3 : Lids 4 : Receiving device 6 : Rotating bed 7 : Permanent magnet #CMT#POLYMERS : #/CMT# Preferred Components: The cell carrier is made of materials on which cells have good adhesion, preferably plasma-treated, smooth polystyrene blanks made of other polymer materials or dense oxide ceramics with a polished surface of Sponceram(RTM: Ceramic carrier discs) type. The cell carriers comprise a screen fabric with a mesh size of 1-2 mm, preferably 0.4-1 mm, or polymers including polystyrene, polyester, polyethylene, polypropylene and other polymers. The rotating bed is made of cell carriers comprising disks with smooth surfaces, alternately arranged discs of smooth material, screen fabric or screen fabric discs. #CMT#EXAMPLE : #/CMT# Mesenchymal stem cells from human bone marrow were isolated and adhered to cell carriers of smooth polymer discs (polystyrene) in the pressure-rotating bed bioreactor. These stem cells were suspended in a medium for the propagation (Dulbecco's modified eagle's medium with addition of human serum). The cell suspension was transferred into a reactor vessel, and a rotating bed comprised cell carriers parallel to the axis of rotation. Then the horizontally oriented cell carrier discs were unmoved until the suspended cells were completely adhered (3-8 hours). Then the rotating bed was put into slow rotation, and the overlay area of the reactor was supplied with controlled mixture of nitrogen and oxygen with pre-selected values of pH and the medium was pumped. A portion of the medium volume was replaced for many days and was respectively continuously fed with fresh medium. The amount of supplied fresh medium was controlled such that the glucose concentration in the reactor was 40% of the glucose concentration in the fresh medium. Under these conditions the differentiation of the cells was unaltered. A growing layer of cells, which were embedded in self-generated extracellular matrix (ECM) was produced on the cell carriers. The further cultivation was continued in the medium to promote differentiation of cells into chondrocytes. The pressure was applied in a phase wise manner such that the reactor chamber was pressurized alternately respectively for 1 hour and was pressure free for 1 hour. On the first day, a pressure of 3 bars was applied and the pressure was increased respectively by 3 bars for following days of cultivation. The initial jelly-like layer of cells and ECM was increasingly firm, which was similar at the end of culturing human cartilage. The cartilaginous, continuous film was obtained from the cell carrier, which had viable cells.</p> |
