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Background: 3D cell culture is an appropriate method to develop engineered bone tissue, where different bioreactors have been designed to mitigate the challenges in 3D culture. Currently, we tailored a perfusion reactor to witness human mesenchymal stem cells (MSCs) proliferation and differentiation over polylactic acid-polyethylene glycol (PLA/PEG) composite scaffolds. Methods: The composite scaffolds with different weight ratios of PLA and PEG were prepared using solvent casting-particulate leaching technique. Human umbilcal card blood MSCs were cultured under dynamic and static conditions to elucidate the role of dynamic fluid flow in osteogenesis of MSCs. Results: The human MSCs distribution over the scaffolds was confirmed with fluorescent microscopy. Alkaline phosphatase (ALP), calcium mineralization, and collagen formation were found to be higher in PLA90 scaffolds than PLA100 and PLA75. PLA90 scaffolds with better cell adhesion/proliferartion were considered for bioreactor studies and they exhibited enhanced ALP, Ca+2 mineralization and collagen formation under dynamic perfusion than static culture. We further confirmed our observation by looking at expression levels of osteogenic marker (Runx2 and osteonectin) in differentiated MSCs subjected to perfusion culture compared to static culture. Conclusion: The results of the current investigation once again proves that dynamic perfusion cultures improve the osteogenic differentiation of MSCs over hybrid polymer scaffolds (PLA90) for effective bone regeneration. Keywords: 3D cell culture, Mesenchymal stem cells (MSCs), Fluid flow, Osteogenic differentiation

Animal cells, beginning mainly as tissue explants, have been cultured outside the body for well over one hundred years. The first culture fluids were simply dilutions of tissue extracts, with their formulation sometimes shrouded in an aura of mystery. During the 1950s basic synthetic culture media, as we know them, composed of many defined primary metabolites and factors, were developed. Yet, for many industrial and pharmaceutical applications animal sera were required for optimal performance. As the needs of culturists, and the understanding of cell biology, have grown so has cell culture media technology. By the turn of this century most biopharmaceutical applications employed commercially prepared and process-optimized medias with protein-free formulations. Exceptions to this included stem cell cultures and many vaccine production formats, many of which continue to this day. Currently, industrial cell culture mainly employs suspension cell cultures in the fed-batch mode with highly optimized animal product-free (and even chemically defined) formulations and feed concentrates, which influences such parameters as product post-translational modifications. Development of new formulations is now occurring for such applications as perfusion culture in intensified batch and continuous biomanufacturing modes, autologous and allogeneic cell therapies, and some 3D culture applications including “bioinks” for 3D bioprinting. Such disciplines as metabolic flux analysis and DoE are now enabling efficient fine-tuning of nutritional requirements and feed timing. Therefore, much of the current development focuses on other growing demands upon media, including business continuity (risk mitigation and supply security), quality (including risk- and science-based approaches to raw material purity), and Regulatory (establishing standards or specific guidance via such organizations as the BPOG).

3D spheroid or organoid culture is becoming mainstream method for studying physiologically relevant cell behaviors, and used in applications such as cell-based diagnostic, therapy, disease modeling and drug screening. Organoids/spheroids function best when maintaining size of

The large-scale production of mammalian cell culture has become one of the most important technologies since the advent of genetic engineering in 1975. Interest in mammalian cell culture intensified with the development of interferons [1]. Suddenly, large amounts of human fibroblasts [2] and lymphocyte cells [3] were needed to run clinical trials and laboratory tests on the so-called “miracle drugs.” The demand for large-scale reactors and systems resulted in rapid gains in the technology. At the same time, culture media, microcarriers [4], and hollow-fiber membranes [5] were also being improved.

This article critically reviews immobilized cells and corresponding bioreactors. Theoretically, cells immobilized with supporting materials are not suitable for the production of primary metabolites, whose production is tightly coupled with cell growth, which could be compromised significantly by the physical confinement of the supporting materials, but applicable for the production of secondary metabolites, whose production is not associated with cell growth, and specialty chemicals via biotransformation with nonvariable or resting cells without the necessity of isolating enzymes at high costs. On the other hand, cells can be immobilized by flocculation or aggregation, which is spontaneous for some species, including microbial, plant, and animal cells, and more convenient for preparation at large scales without extra costs. As no supporting materials are needed, their impact on cell growth can be avoided. Thus, this strategy is suitable for the production of primary metabolites, particularly bulk commodities such as fuel ethanol, scientifically and economically as well. Moreover, the encapsulation of cells for biomedical use to work as in vivo drug factories as well as membrane bioreactors for the production of biologics through the perfusion culture of mammalian cells are revisited from a cell immobilization point of view.

Based on the control scheme of nutrients and metabolites, there are many modes of cell culture processes, including batch, repeated-batch, fed-batch, chemostat, and perfusion, among which batch, fed-batch, and perfusion modes are used for industrial-scale biologics production. Batch is the simplest with little operation control of nutrients and metabolites, whereas perfusion is the most complex with intensive control. Generally, high-level control leads to high productivity, but with a tradeoff of high complexity, increasing risk of failure, and developmental time and cost. The choice of the culture mode is largely dependent on process capacity and quality requirements. Different culture modes are well distinguishable in cell growth, nutrient consumption, and product formation kinetics. Understanding the kinetics is the starting point for process development and improvement. The development of more sophisticated fed-batch and perfusion cultures is rooted in the understanding of the process established in batch culture. Batch culture is the indispensable first step for establishing a proper cell maintenance schedule and for further developing other culture modes with more complexity and higher performance. This article provides detailed discussions on different culture modes and associated culture kinetics and process design and operation considerations in the biotechnology industry.

A hybridoma cell line was cultivated in a new perfusion system using serum- and protein-free medium for more than 800 h. The system consits of an airlift loop reactor (volume 5.4 1) and a lamellar clarifier (volume 1.7 1) in a by-pass stream. Free flow convection through the settler is achieved by cooling the cell suspension before entering the settler. Thus no mechanical pumping is required. In comparison to a simple settler of the type “Dortmund Brunnen” (volume 2.4 1) the perfusion rate could be increased from 1.45 to 4 reactor volume/d. Retention efficiency for viable cells is about 90 % whereas dead cells are washed out preferably.

A perfusion bioreactor configuration is presented which incorporates a conical lamella settler within a reactor vessel which is itself conical. The method used in determining dimensions for the sedimenter and in scaling up the apparatus ensures that cell residence time in the seperator is independent of reactor working volume. A benchtop-scale prototype of the reactor was tested with hybridoma cells. A 30-day perfusion culture in this vessel reached a maximum cell concentration of 6.5 million cells/mL, with a specific antibody production rate four times that seen in batch culture.

A new kind of estimator based on a general kinetic model of mammalian cell cultures has been developed. When using the measurements of ammonia and LDH in the culture medium, the estimator predicts the time variation of the viable and dead cells, glucose, glutamine, lactate and antibodies concentrations during a hybridoma perfusion culture.

The high density culture of recombinant CHO was carried out with both Cytodex 1 and Asahi Microcarrier which is a new macroporous microcarrier made of cellulose. The perfusion culture vessel was a 500 ml spinner glass flask with a sedimentation column for separation. For supplying oxygen, a Teflon porous tube coil was connected in the flask. Both cultures were perfused for 20 days. When using Cytodex 1, the cell density reached 5 × 106 cells/ml and the M-CSF production during 20 days was 6.6 mg. However, when using Asahi Microcarrier, the cell density reached 1.5 × 107 cells/ml and the M-CSF production during 20 days was 48.0 mg. The high density culture and an effective production were successfully achieved with the new macroporous microcarrier.

Perfusion of cultures of suspended recombinant Chinese hamster ovary (CHO) cells was accomplished through the use of an external, 3-port inclined settler which was shown to preferentially retain viable cells in the bioreactor. By vibrating the settler and cooling it to 4°C, an average of 89% of viable cells were returned to the bioreactor while the return of nonviable cells was only 72%, with the rejected cell population being only 36% viable when settler feed contained 78% viable cells. The residence time of CHO cells in the cooled, vibrated settler was determined by flow cytometric discrimination of tracer recombinant CHO cells. Cells returning to the bioreactor through the underflow had an average residence time of 1.46 hours in the settler. During perfusion cultures with cell densities above 106 cells/mL, cells seen to be stalled within the settler were easily dislodged by periodic air bubbling using a simple backflushing procedure in which headspace gas was brought through the settler underflow port. 87% of the nonretuming cells, which represented about 25% of the cells which settled, were returned to the bioreactor after bubbling within an average of 32 minutes after bubbling.

The production of a recombinant protein was assayed in a new Mamestra brassicae cell line grown at high density in a serum free medium. The gene coding for Drosophila melanogaster acetylcholinesterase (AchE) was inserted in a baculovirus under the control of the P10 promoter. Cell growth rate and metabolism, together with the rate of production of the recombinant protein, were comparatively measured in a standard stirred bioreactor and in a perfused stirred bioreactor coupled to an external cell recycling system (the Cytoflow of SGI). In the perfused culture the cell growth was linear and reached a maximum cell density of 5×107 cells per ml, with a stable cell viability near 90% in 13 days. Cell concentration was ten fold the concentration observed in batch. Compared to the batch culture, total yield of AchE in the perfused culture was 13-fold higher within a period time that was only twice. The perfusion culture increased the cell specific productivity 8.6 fold. In terms of medium cost, the perfusion rate used in these experiments never exceeded 2.7 working volume per day.

Hybridoma cells have been grown in a dialysis perfusion system, with continuous replacement of the dialysed medium. Cell densities of 2.5 × 10 6 cells/ml have been maintained for 10 days in a steady state, which has been shown to be the result of a balance between continuing growth and death of the retained cells. Total antibody content reached 1.44 g after 18 days in the 1l vessel. The specific yield on basal medium was twice that in a typical batch culture, and the specific yield on serum used was 19 times higher than in either batch culture, or other perfusion systems. Cells continued to synthesise DNA as shown by 3 H-Thymidine incorporation during the steady state, which reflected the balance between cell growth and cell death. Fluorescence activated cell sorting (FACS) showed the presence of a low intracellular antibody subpopulation which disappeared over the course of the fermentation. KEYWORDS Perfusion culture, hybridoma, monoclonal antibody, high density culture.

A simple perfusion culture of a murine hybridoma is described, that produced 750 mg of lgG1 over 25 days. Features of the experiment were low cell viabilities and a slowly increasing lactate:glucose ratio.

A continuous flow centrifuge has been used as a perfusion system for high density culture of hybridomas and HeLa cells in stirred tank bioreactors. During continuous runs of two weeks no clogging of cells inside the rotor could be observed. More than 99.99% of the cells could be retained and recirculated into the bioreactors. Cell viability was not influenced by the centrifugation process or circulation by peristaltic pumps. The cell specific productivity of hybridoma cells was significantly increased compared to batch culture. Keywords: Centrifuge, continuous flow centrifugation, high density culture, perfusion culture

Porous collagen microspheres have been developed which are suitable for both attachment-dependent cells and suspension cells. Key features of this macroporous microsphere cell culture in a fluidized-bed bioreactor are the increased matrix surface area and open volume resulting in high cell density and efficient mass transfer of nutrients and transport of secreted products into the harvest. Results have shown that this microsphere fluidized-bed culture is extremely suitable for the culture of all types of hybridoma cells as well as for recombinant and naturally secreting mammalian cells. The results from a fluidized-bed bioreactor culture of an anti-interleukin-6 (IL-6) producing hybridoma are presented. This bioreactor culture with a fluidized-bed volume of 0.7 L was initiated in culture medium, supplemented with 1% fetal bovine serum (FBS). On Day 11 and thereafter, culture medium containing insulin as the sole protein was perfused for 38 days. Under steady-state conditions, this bioreactor produced an average of 45 mg/L of anti-IL-6 antibody with a daily medium perfusion rate of 17 L, thus resulting in daily production of > 1 g antibody per L of fluidized bed. Per cell productivity of this culture was about 2-fold higher than the batch culture. SDS-PAGE analysis (reduced) of the reactor harvest samples showed the presence of only two protein bands, i.e., one for the IgG heavy chain and one for its light chain illustrating that the IgG purity of the raw culture harvest was exceedingly high. Results from a second hybridoma cell line which produces antibody specific for murine IL-3 show a similar increase in per cell productivity. The reasons for the production of high quality MAbs and recombinant proteins in this culture process are discussed.

A perfusion culture system has been developed which utilizes a porous ceramic foam for the cultivation of adherence-dependent cells. The cells grow within the interconnected pores of a rigid, reticulated, alumina foam matrix. Concentrations of BHK cells in excess of 108 per ml of matrix volume have been achieved. Stable expression of human transferrin in these cells was maintained at high levels (2.4 μg 106 cell–1 day–1) for a period of greater than 4 weeks. Vero cells grown in the matrix could be recovered readily by trypsinization making this system suitable for the large-scale preparation of inocula for other cultivation systems such as conventional or porous microcarrier cultures. The matrix can be installed in stirred tank or air lift reactors and is amenable to scale-up.

Mouse-human hybridoma X32 cells were cultivated using a perfusion culture apparatus provided with three settling zones where the cells were separated from the culture medium by gravitational settling, as oxygen was supplied using a perfluorocarbon. Viable cell density reached about 2 × 10 cells·ml–1 at 2.0 vol·vol–1·d–1 specific perfusion rate, and the monoclonal antibody was continuously produced. This viable cell density was significantly higher than in the perfusion culture of a gravitational settling type provided with one settling zone, as oxygen was directly sparged (about 1 × 107 cell·ml–1).