Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system

Abstract

To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET(®) tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written.

Keywords: 3D culture; Biomaterial testing; Biomedicine; Bioreactor; Cell culture; Perfusion culture; Tissue carrier; Tissue engineering.

Fig. 1

Application of a MINUSHEET^® tissue carrier. a First a biomaterial measuring 13 mm in diameter is selected. b Then the biomaterial is placed in the black base part of a tissue carrier. Mounting is completed by pressing the white tension ring in the base part. c After sterilization the carrier is transferred by forceps to a 24 well culture plate for cell seeding

Fig. 2

Versatile use of a MINUSHEET^® tissue carrier in a perfusion culture container. a In a basic version a perfusion culture container can hold six tissue carriers for provision with always fresh medium. b In a gradient perfusion culture container the contained tissue is exposed to different fluids at the luminal and basal side. c For observation of growing tissue under a microscope a transparent lid and base is integrated in a container. d A perfusion culture set up is running in the typical case on a laboratory table and under atmospheric air. A thermo plate maintains the desired temperature of 37 °C. During culture a peristaltic pump transports the medium (1.25 ml/h) from a storage bottle (left side) to the waste bottle (right side). Arrow indicates flow of medium

Fig. 3

Features of a renal collecting duct (CD) epithelium kept for 13 days in a gradient perfusion culture container. At the luminal side IMDM + aldosterone (1 × 10⁻⁷ M) + 15 mmol/l NaCl, while at the basal side IMDM + aldosterone (1 × 10⁻⁷ M) was transported. Immunohistochemistry shows that an intense label for tissue-specific markers such as a TROMA I, b cingulin and c Na/K ATPase α5 is present. Site of the basal lamina is marked by an asterisk, while lumen is indicated by an arrow

Fig. 4

Generation of renal tubules at the interface of a polyester interstitium after 13 days by perfusion culture. a Scanning electron microscopy demonstrates development of numerous renal tubules (T). b Fluorescent label for Soybean Agglutinin shows numerous tubules developing within an artificial interstitium. c Semithin section after Richardson staining shows generated tubules in oblique, respectively, vertical view between fibers of the polyester fleece (PF). Site of the basal lamina is marked by an asterisk, while lumen is indicated by an arrow

Fig. 5

Transmission electron microscopy demonstrates renal tubules generated at the interface of a polyester interstitium after 13 days of perfusion culture in a Iscove’s Modified Dulbecco’s Medium, b CO₂ Independent Medium and c Leibovitz’s L-15 Medium. In all cases generated tubules exhibit a polarized epithelium. Neighboring cells are separated by a tight junction complex (arrow head). The basal lamina is indicated by an asterisk