Industrial choices for protein production by large-scale cell culture.

Substantial progress made in the areas of stem cell research and regenerative medicine has provided a number of innovative methods to repair or regenerate defective tissues and organs. Although previous studies regarding regenerative medicine, especially those involving induced pluripotent stem cells, have been actively promoted in the past decade, there remain some challenges that need to be addressed in order to enable clinical applications. Designed for use in clinical applications, cell sheet engineering has been developed as a unique, scaffold-free method of cell processing utilizing temperature-responsive cell culture vessels. Clinical studies using cell sheets have shown positive outcomes and will be translated into clinical practice in the near future. However, several challenges stand in the way of the industrialization of cell sheet products and the widespread acceptance of regenerative medicine based on cell sheet engineering. This review describes current strategies geared towards the realization of the regenerative medicine approach.

Cell sheet engineering for regenerative medicine: current challenges and strategies.

The ability to create DNA double-strand breaks (DSBs) at specified genomic locations, which then stimulate the cell’s naturally occurring DNA repair processes, has introduced intriguing possibilities for genetic modification. Zinc finger nucleases (ZFNs) are designed restriction enzymes consisting of a nonspecific cleavage domain fused to sequence-specific DNA binding domains. ZFN-mediated DSB formation at endogenous genomic loci followed by error-prone non-homologous end joining (NHEJ) repair can result in gene-specific mutations via nucleotide base pair insertions or deletions. Similarly, specific DNA sequence modifications can be made by providing donor DNA templates homologous to sequences flanking the cleavage site via homology-directed repair (HDR). Targeted deletions of intervening DNA sequence can be obtained by ZFNs used to create concurrent DSBs. Site-specific transgene integration into ZFN-induced DSBs is possible via either NHEJ or HDR. Genome editing can be used to enhance our basic understanding of plant gene function as well as modify and improve crop plants. As with conventional plant transformation technology, the efficiency of genome editing is absolutely dependent on the ability to initiate, maintain, and regenerate plant cell and tissue cultures.

/pdf/genome-editing-in-plants-via-designed-zinc-finger-nucleases-bg2tyeuflf.pdf

Genome editing in plants via designed zinc finger nucleases.

This paper reviews early studies of regenerative medicine using human cells and engineered tissues progressing from a laboratory-centered manual procedure toward automated manufacture. It then examines the distinctive bioprocesses by which autologous human material must be produced, the degree of simplification allowed by use of allogeneic cell lines and engineered tissue derived from them, and issues that affect both cell types. The paper concludes by drawing upon this discussion to suggest some factors that will determine how regenerative medicine bioprocessing can progress to provide many units of material economically.

Regenerative medicine bioprocessing: building a conceptual framework based on early studies.

Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique.

The robot automation of sampling and the subsequent treatment and storage of aliquots during mammalian cell cultivations was investigated. The complete setup, the development and testing of the sampling device, the robot arm, and the cell imaging system are described. The developed sampling device is directly coupled to a pilot bioreactor. It allows the computerized sterile filling of cell broth into 50 mL sample tubes. After each sampling the whole tubing system is steam sterilized. For further off-line treatment a robot takes the sample to the different devices. This robot is equipped with a camera and a force/torque sensor. A color-based object recognition guides the arm in a complex surrounding with different illumination situations, enabling the robot to load the sampling device with tubes and take the sample to further devices. For necessary pipetting and refilling we developed a computerized device. Cells are automatically stained and counted using an imaging system. The cell number and viability is automatically saved in a process control system together with the on-line parameters. During several cultivations in 20 and 100 L scale these main components of the automation strategy were successfully tested.

First steps in robot automation of sampling and sample management during cultivation of mammalian cells in pilot scale.

In this paper we present a mobile robot system that is capable of automating the sample management in a biotechnological laboratory. The system consists of a mobile platform and a robot arm. It can navigate freely in the laboratory and operate standard devices needed for the sample management. The platform uses an extended Kalman filter for localization and A* algorithm for path planning on a tangent graph computed from the laboratory's map. Motion execution has been designed to be as predictable as possible to not irritate, disturb or harm human personnel. The robot arm uses color vision to detect devices and compensate for positioning errors. The parameters and tasks needed to operate the devices are specified in simple scripts to allow quick and easy adaptations to other situations.

/pdf/a-service-robot-for-automating-the-sample-management-in-32q0vxakjr.pdf

A service robot for automating the sample management in biotechnological cell cultivations

On inflamed endothelium the cell surface protein E-selectin isexpressed which supports the initial process of attachment –capturing and rolling of leukocytes A recombinant CHO cell linesecreting a soluble E-selectin-IgG chimera was cultivated competitively under serum free conditions in three different bioreactor systems: a 1 l Super-Spinner, a 2 l stirred tank bioreactor equipped with a spinfilter, and a 100 l stirred tankbioreactor In the smallest system 254 mg E-selectin-IgG wereproduced in 62 days using a repeated batch process whileachieving a maximal viable cell density of 37 × 106 cells ml-1 Using continuous perfusion mode a total amount of352 mg were produced with a maximal viable cell density of165 × 107 cells ml-1 in the 2 l bioreactor within 29 days Large scale cultivation in a 100 l stirred tankbioreactor yielded 1056 mg in three batches with a maximal viable cell density of 97 × 105 cells ml-1 within 15 days After removal of the cells by continuous centrifugation and a depth filter clearance step, the supernatants were concentrated via ultra filtration Purificationwas performed by affinity chromatography with rProtein A Integrity of the E-selectin-IgG protein was checked with SDS PAGE Its activity was verified in a cellular adhesion assay performed with HL-60 cells and a recombinant CHO cell line expressing membrane-anchored E-selectin constitutively, and E-selectin expressing HUVECs, respectively Soluble E-selectin-IgG was used to block adhesion to these cell layerscompetitively A concentation of 188 and 375 μg ml-1was sufficient to reduce the amount of adhering HL-60 cells to 50% on CHO and HUVEC layers, respectively

/pdf/serum-free-production-of-a-chimeric-e-selectin-igg-protein-3nb7ttqiaa.pdf

Serum-free production of a chimeric E-selectin-IgG protein from 1 to 100 l scale: Repeated batch cultivation versus continuous spin filter perfusion

Robot Automation of Sampling and Sample Management During Cultivation of Mammalian Cells on Pilot Scale

In mammalian cell culture technology the last key issue for a completely automated process is the taking, analysing and storing of a sample from the bioreactor. It was necessary to develop a new device which fulfils the need to be steam sterilizable for an application in industrial production environment. The status of the culture is derived from the results of the analysed parameters of these samples. The normal procedure includes determination of the total and viable cell densities in relation to the media parameters (glucose, lactic acid, ammonia). The cell densities are retrieved by counting a stained cell sample. A cell free aliquot is stored in a freezer for further analyses (e.g. amino acids and product). Information for optimisation of feed and harvest strategies can be retrieved from the analysed parameters additionally.

Robot Automation of Sampling and Sample Management during Cultivation of Mammalian Cells in Pilot Scale

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