Perfusion Culture Container Research Articles

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

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.

Mimicking the Bone Marrow Niche in Vitro: Establishment of a 3D Perfusion Culture System for Mesenchymal Stromal Cells

IntroductionHematopoiesis takes place in the bone marrow niche. The niche is created by a specific interplay of different cell types and signalling molecules such as growth factors and cytokines. The self-renewal and survival of hematopoietic stem cells (HSCs) as well as their proliferation and differentiation are stimulated by these microenvironmental factors. Hematological disorders such as leukemia are associated with disruptions of the microenvironment within the bone marrow niche. To get further insights into the interplay of cells and molecules creating the niche and potential alterations due to hematological disorders, it is of importance to develop a model which mimics the niche in vitro. Thus, the aim of this study was to establish a 3D perfusion culture system for human mesenchymal stromal cells (MSCs), which constitute an important supportive cellular component of the bone marrow niche.MethodsWe used a modular perfusion culture system (Will W. Minuth, Regensburg, Germany). The system was already applied for culturing of a variety of different tissues, but not yet for culturing human MSCs.MSCs were cultured on porous membrane filters consisting of mixed cellulose esters (pore size: 0.45 µm). Membranes were placed into a container, which was permanently perfused with fresh culture medium. Thus, the cells were constantly provided with nutrition while the accumulation of toxic metabolic products was prevented.To establish the system, several parameters were varied in order to find out optimal conditions for the MSCs in perfusion culture. The influence of the following parameters was analyzed: the material of the membranes, the cell seeding volume, the position of the membranes in the perfusion culture container, the concentration of HEPES buffer in the medium and its flow rate. The viability of the MSCs in different culture conditions was tested by applying an MTS assay. Additionally, morphology of MSCs and the expression of exemplary selected genes important for the bone marrow niche (CXCL12 and JAG1) were analyzed. MSCs cultured under conventional cell culture conditions served as controls. Statistical analysis was performed by using the Student’s t-test (GraphPad Prism 6).ResultsWe could show that the amount of viable MSCs cultured under ideal conditions in perfusion culture was considerably higher than in conventional cell cuture (OD450: 0.37 ± 0.03 vs. 0.2 ± 0.01, P-value < 0.01 %). A perfusion rate of 20.8 µL per minute and a HEPES concentration of 50 mM were observed to be optimal for the viability and growth of the MSCs. The cells showed no differences in gene expression levels due to the different culture conditions.Table 1:Relative quantification of CXCL12 and JAG1 mRNA levels in MSCsDeltaCTCXCL12DeltaCTJAG1Perfusion culture8.4025.967Conventional cell culture8.0086.750Additionally, the MSCs cultured on membranes formed 3D-like networks. In perfusion culture, the MSCs seemed to grow in a more orderly manner compared to conventional cell culture conditions. No differences in morphology were observed due to the different culture conditions.DiscussionThe 3D perfusion culture system is sufficient to increase the viability and the growth of the MSCs without changing the gene expression profile of exemplary chosen genes relevant for homing and adhesion of HSCs in the bone marrow niche. The morphology of MSCs also did not change due to the different culture conditions.ConclusionThis system can be used for further experiments including co-culturing experiments with MSCs and HSCs and/ or leukemic blasts and might be an important option to mimic the hematopoietic stem cell niche in vitro. DisclosuresNo relevant conflicts of interest to declare.

Supportive development of functional tissues for biomedical research using the MINUSHEET® perfusion system

Functional tissues generated under in vitro conditions are urgently needed in biomedical research. However, the engineering of tissues is rather difficult, since their development is influenced by numerous parameters. In consequence, a versatile culture system was developed to respond the unmet needs.Optimal adhesion for cells in this system is reached by the selection of individual biomaterials. To protect cells during handling and culture, the biomaterial is mounted onto a MINUSHEET® tissue carrier. While adherence of cells takes place in the static environment of a 24 well culture plate, generation of tissues is accomplished in one of several available perfusion culture containers. In the basic version a continuous flow of always fresh culture medium is provided to the developing tissue. In a gradient perfusion culture container epithelia are exposed to different fluids at the luminal and basal sides. Another special container with a transparent lid and base enables microscopic visualization of ongoing tissue development. A further container exhibits a flexible silicone lid to apply force onto the developing tissue thereby mimicking mechanical load that is required for developing connective and muscular tissue. Finally, stem/progenitor cells are kept at the interface of an artificial polyester interstitium within a perfusion culture container offering for example an optimal environment for the spatial development of renal tubules.The system presented here was evaluated by various research groups. As a result a variety of publications including most interesting applications were published. In the present paper these data were reviewed and analyzed. All of the results point out that the cell biological profile of engineered tissues can be strongly improved, when the introduced perfusion culture technique is applied in combination with specific biomaterials supporting primary adhesion of cells.

Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Objective To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system. Methods Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using 35SO 4 2−-labelling and the dimethylmethylene blue assay. 3H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured. Results The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days. 3H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the 35SO 4 2− incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts. Conclusion The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.

A modular culture system for the generation of multiple specialized tissues

Numerous factors influence cell functions and tissue development in culture. A modular culture system has been developed to allow the control of many of these important environmental parameters. Optimal adhesion of cells is obtained by selecting an individual biomaterial. Selected specimens are mounted in a tissue carrier in order to protect it against damage during handling and after seeding cells, the carriers can be used in a series of compatible perfusion culture containers. This technique allows the simple bathing of growing tissue under continuous medium transport and the exposure of epithelia to a gradient with different fluids at the luminal and basal sides. A further container is made of transparent material to observe microscopically the developing tissue. In addition, a special model features a flexible silicone lid to apply force to mimic the mechanical load required for developing connective and muscular tissue. Perfusion culture of stem/progenitor cells at the interface of an artificial interstitium made by a polyester fleece results in the spatial development of tubules. During long term culture over weeks the growing tissue is continuously exposed to fresh nutrition and respiratory gas. The medium is transported in a constant flow or in pulses, preventing unstirred layers of fluid. A variety of applications of this modular system, described in this paper, demonstrates that the biological profile of cells and tissues can be strongly improved when perfusion culture with a permanent provision of fresh medium is applied.

The formation of pores in the basal lamina of regenerated renal tubules

Little information is available concerning the generation of renal tubules, but this information is urgently needed in regenerative medicine for the future treatment of acute and chronic renal failures. Of major interests are the integration of stem/progenitor cells, the cellular development and the tubular growth in a spatial environment. In this regard, we investigated the basal aspect of renal tubules generated at the interphase of an artificial interstitium. Stem/progenitor cells derived from neonatal rabbit kidney were mounted inside a specific tissue holder and covered by layers of polyester fleece. The tissue was then kept in a perfusion culture container for 13 days in chemically defined IMDM containing aldosterone (1 × 10 −7 m) as a tubulogenic factor. The spatial development of tubules was registered on whole-mount specimens and on cryo-sections labeled with soybean agglutinin (SBA) and tissue-specific antibodies indicating that collecting duct tubules were developed. Scanning electron microscopy (SEM) revealed that the generated tubules were completely covered by a basal lamina. Most interestingly, the matrix was not consistently composed, but exhibited three categories of pores. The most frequently found pore type had an apparent diameter of 133 ± 26 nm followed by a medium-sized pore type of 317 ± 35 nm. Another category of pores with a diameter of 605 ± 101 nm was rather rarely found. All of the pores were evenly distributed and not restricted to particular sites. The newly detected pores are not related to culture artifacts, since they were also detected in collecting duct tubules of the neonatal rabbit kidney. It remains to be evaluated whether these pores support physiological transport functions or if they indicate the site where extracellular matrix proteins are inserted into newly synthesized basal lamina.

The tubulogenic effect of aldosterone is attributed to intact binding and intracellular response of the mineralocorticoid receptor

Abstract Little is known about the extra- and intracellular stimuli inducing renal stem/progenitor cells to develop into three-dimensionally structured tubules. To study this specific development in a controlled environment, we used an advanced culture technique. Embryonic tissue derived from neonatal rabbit kidney was placed in a perfusion culture container at the interface of an artificial interstitium made of a polyester fleece. Culture was carried out in chemically defined Iscove’s Modified Dulbecco’s Medium (IMDM) for 13 days. Development of tubules was histochemically detected on cryosections labeled with Soybean Agglutinin (SBA). The experiments showed that aldosterone exerts a specific tubulogenic effect. Application of aldosterone (1 × 10−7 M) raised numerous SBA-labeled tubules, while in the absence of the steroid hormone the development of tubules was lacking. Specificity of hormone action was analyzed by the use of aldosterone antagonists. Administration of spironolactone (1 × 10−4 M) and canrenoate (1 × 10−5 M) completely inhibited the development of tubules. Finally, disrupting the intracellular molecular complex of the mineralocorticoid receptor (MR) and heat shock proteins by geldanamycin (2 μg/ml) prevented the development of tubules. Our results suggest that the tubulogenic effect induced by aldosterone is attributed to both hormone binding and an undisturbed intracellular response of the MR.

Chemically defined medium environment for the development of renal stem cells into tubules

The use of stem cells is a valuable therapeutical option for the regeneration of diseased tissues and organs. However, the involved cellular processes are hardly known. To gain detailed information about their development, a new culture technology was developed. Embryonic renal tissue containing stem/progenitor cells was mounted within a perfusion culture container at the interface of an artificial interstitium made of polyester. Using this innovative approach we show that renal tubules develop in chemically defined Iscove's modified Dulbecco's medium without serum addition and without coating by extracellular matrix proteins. The development of tubules depends on the administration of aldosterone, and can be visualized by immunohistochemical labeling. The presented technology makes the exact analysis of developmental steps now possible, and provides a new powerful tool to optimize growth and differentiation of renal stem cells. It may also enable many other kinds of stem cells to steer their development into functional tissues under clearly defined in vitro conditions.

The role of polyester interstitium and aldosterone during structural development of renal tubules in serum-free medium

Little knowledge is available regarding the development of renal stem/progenitor cells into functional parenchyme. To investigate the environmental mechanisms during this maturation process, we elaborated an advanced culture technique to follow renal tubule development. Embryonic stem/progenitor cells derived from neonatal rabbit kidney were placed in a perfusion culture container at the interphase of an artificial polyester interstitium. Tissue culture was carried out in IMDM without serum or protein supplementation and without coating with extracellular matrix proteins. Development of tubules was registered histochemically on cryosections labeled with soybean agglutinin (SBA) and tissue-specific antibodies. The experiments revealed that the development of renal tubules depends exclusively on the administration of aldosterone. The use of 1×10 −7 m aldosterone for 13 days generated numerous SBA-labeled tubules, while no tubules developed in the absence of the steroid hormone. To obtain further information about the action of the hormone on the cognate receptor, molecular precursors of the aldosterone synthesis pathway were tested. Surprisingly, application of cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (DOCA) and corticosterone failed to form numerous tubules. Only 11-DOCA and progesterone induced a few tubules, which were barely SBA-labeled. Furthermore, application of aldosterone antagonists such as 1×10 −4 m spironolactone and 1×10 −4 m canrenoate completely inhibited the development of tubules. We conclude that specifically aldosterone promotes the development of tubules via the mineralocorticoid receptor whereas its precursors have no effect.

Modulating the Development of Renal Tubules Growing in Serum-Free Culture Medium at an Artificial Interstitium

Little information on the structural growth of renal tubules is available. A major problem is the technical limitation of culturing intact differentiated tubules over prolonged periods of time. Consequently, we developed an advanced culture method to follow tubule development. Isolated tissue containing renal progenitor cells was placed in a perfusion culture container at the interphase of an artificial polyester interstitium. Iscove's modified Dulbecco's medium without serum or protein supplementation was used for culture, and the culture period was 13 days. Tissue growth was not supported by addition of extracellular matrix proteins. The development of tubules was registered on cryosections labeled with soybean agglutinin (SBA) and tissue-specific antibodies. Multiple SBA-labeled tubules were found when aldosterone was added to the culture medium. In contrast, culture without aldosterone supplementation displayed completely disintegrated tissue. The development of tubules depended on the applied aldosterone concentration. The use of 1 x 10(-6) M and 1 x 10(-7) M aldosterone produced numerous tubules, while application of 1 x 10(-8) M to 1 x 10(-10) M led to a continuous decrease and finally a loss of tubule formation. The development of labeled tubules in aldosterone-treated specimens took an unexpectedly long period of at least 8 days. The morphogenic effect of aldosterone appeared to be mineralocorticoid hormone-specific since spironolactone and canrenoate abolished the development. Finally, dexamethasone induced widely distributed cell clusters instead of tubules.

Growth of embryonic renal parenchyme at the interphase of a polyester artificial interstitium

The construction of an artificial kidney module by tissue engineering or the application of cell-based therapies for the treatment of renal failure requires exact information regarding the cellbiological mechanisms of parenchyme development in combination with different kinds of biomaterials. To learn more about these processes tissue cultures are frequently used experimental tools. However, apart from experiments with early kidney anlagen there is a lack of suitable in-vitro models regarding the generation and long-term maintenance of renal tubules. In the present paper we like to demonstrate an advanced culture technique, which allows to generate tubular elements derived from renal stem cells. For the growth of tubules it is essential to fine-tune the interface between the embryonic tissue and the dead fluid space within a perfusion culture container by offering a polyester artificial interstitium. Culture was performed in IMDM supplemented with hormones and growth factors but using serum-free conditions over 14 days. Formation of tissue was then analysed by immunohistochemistry and two-dimensional (2D) electrophoresis. Culture in pure IMDM leads to a complete loss of tissue formation. In contrast, application of aldosterone (A) induces the development of numerous polarised tubules. Surprisingly, addition of epidermal growth factor (EGF), a cocktail of insulin, transferrin and selenium (ITS), retinoic acid (RA), cholecalciferol (VitD 3) or bovine pituitary extract (BPT) does not further improve development of tubules, but leads to intensive cell clustering and a decrease of tubule formation. 2D Western blots of developing tissue probed with soybean agglutinin (SBA) reveal a unique pattern of newly detected proteins. It is found that growth factors do not support but abolish protein spots upregulated by aldosterone. It remains to be investigated, which cellbiological effect stimulates the embryonic cells to develop tubules in competition to cell clusters at the interphase of an artificial interstitium.

Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.