Method and device for cell modification

a cell modification and cell technology, applied in the field of cell modification, can solve the problems of difficult culturing large numbers of cells adhered difficult to achieve culturing large numbers of cells to adhere to a surface without the use of difficult to achieve culturing large numbers of cells to adhere to a surface without the use of carriers or large volume cell suspensions, etc., to achieve speed up the development o

Inactive Publication Date: 2014-10-23
MILTENYI BIOTEC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0007]The effect of enhanced gravity generated by centrifugation on cells under culturing conditions has been investigated in various publications. Huang et al (2009) disclose in “Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells” in J. Biomed. Here, rBMSCs are first plated on glass coverslips; after 24 h the cells had adhered to the coverslips and the coverslips were transferred to a biocompatible polyethylene culture bag, are incubated with medium and then cultured on a cell centrifuge at 2 g hypergravity for several days. The medium was changed every 3 days during HG/SMG culture.
[0008]Gaubin et al. described in Microgravity Sci. Technol. 1991 February; 3(4):246-50 the effects of hypergravity on adherent human cells. Galimberti et al disclose in “Hypergravity speeds up the development...

Problems solved by technology

Slow macroscopic convection of the medium results in uncontrolled and uneven supply of nutrients to the cells and may result in different differentiated i.e. manipulated cells.
Culturing large numbers of cells adhered to a surface without the use of carriers or large volume cell suspension is difficult and requires frequent change of the medium.
The known static systems for cell culturing are labor-intensive and need clean room conditions during handling the cell cultures, for example media exchange or transf...

Method used

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  • Method and device for cell modification
  • Method and device for cell modification

Examples

Experimental program
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example 1

Viral Transduction of T Cells with Disease-Specific T Cell Receptor Genes

[0167]A use of the invention is the introduction of genes coding for a disease-specific T cell receptor into a polyclonal population of T cells, which may then be used for therapeutic injection into patients. The T cells are directed towards the target antigen, e.g. tumor cell or infected cells.

[0168]A centrifugation chamber providing cell modifying surfaces coated with RetroNectin® is supplied with a recombinant virus containing supernatant, wherein the virus encodes the target antigen, and rotated at surfaces by the gravitational forces generated by the rotation. Following this coating step, the chamber is rotated at low rotation speed and the T cells to be modified are introduced into the high rotational speed (e.g. 2000×g) for 2 hours. For improved viral transduction, the T cells are previously activated, e.g. by cultivation in the presence of antibodies against CD3 and CD28, either in the same centrifugati...

example 2

Activation and Expansion of Antigen-Specific T Cells

[0171]T cells can be activated and expanded by antigens loaded in or on antigen-presenting cells (APC). T cell activation requires intimate contact between the T cells and APC.

[0172]To improve T cell activation a system described herein is used to spin down APC and T cells in an appropriate ratio, e.g. 1:100 to 100:1. Either physiological cell mixtures such as PBMC, containing T cell and APC or defined cell preparations, e.g. purified T cells and APC, e.g. dendritic cells, B cells, macrophages, cell lines transfected with distinct MHC molecules, etc, mixed at an appropriate ratio are used. In addition antigens, proteins, peptides, cell lysates, and growth factors and / or co-stimulatory antibodies, e.g. anti CD28, antiCD137, may be added. The contact between the cells is rapidly induced and maintained at an appropriate level by centrifugation.

[0173]APC and T cells can be deposited in distinct layers, e.g. T cell on top of a layer of ...

example 3

Polyclonal Activation and Expansion of T Cells

[0176]The systems of the invention provide an optimized platform for polyclonal activation and expansion of T cells, comprising conventional T cells or regulatory T cells.

[0177]This example is similar to Example 2 except that instead of defined antigen, polyclonal stimuli are used, comprising antibodies against CD3 and co-stimulatory molecules, such as CD28 and / or CD137. These antibodies are added either in soluble form, requiring the addition of accessory cells bearing Fc-receptors, e.g. conventional antigen-presenting cells or cell lines transfected with Fc-receptors. Alternatively the added antibodies are immobilised on a macroscopic surface, e.g. a particle or bead ranging from about 30 nm to 100 μm. These immobilised antibodies are directly cultured with purified T cells, e.g. at ratios 1:4 to 4:1. As described above the system used allows regulated contact of T cells and stimulating agent and controlled addition of additional envir...

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Abstract

The invention relates to a cell modification device, comprising a centrifugation chamber with at least one cell modifying surface with a normal vector having an angle of 135-45° to the rotational axis of the centrifugation chamber, wherein the centrifugation chamber comprises at least one input/output port and the cells to be modified are immobilized at the cell modifying surfaces by the rotation of the centrifugation chamber at 2 to 2000 g. Furthermore, the invention relates to a method for modifying cells comprising the steps—introducing cells in a cell modification device, comprising a centrifugation chamber with at least one cell modifying surface with a normal vector having an angle of 135-45° to the rotational axis of the centrifugation chamber wherein and comprising at least one input/output port, —immobilizing the cells on the cell modifying surfaces by the rotation of the centrifugation chamber at 2 to 2000 g—maintaining the rotation of the rotation of the centrifugation chamber until the cells are modified.

Description

DISCLOSURE OF INVENTION[0001]The present invention relates to methods and devices for modifying eukaryotic cells on functionalized surfaces of a centrifugation apparatusBACKGROUND OF THE INVENTION[0002]The conditions during cell culturing have a substantial impact on the phenotypes of the cells and desired or not, cell culturing leads to the manipulation of cells.[0003]Cell culture refers to methods under which eukaryotic cells, especially of mammalian origin, are maintained at appropriate conditions with supply of cell culture medium in a cell incubator or a fermenter. Cell culture conditions vary widely depending on the cell type and the desired application. Variation of cell culture conditions can be utilized for cell expansion, cell differentiation or manufacturing of different phenotypes of the cell type. The most commonly varied factor in culture systems is the cell culture medium, for which a vast number of recipes is known (see for example “Cell Culture Techniques” Humana Pr...

Claims

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Application Information

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IPC IPC(8): C12N15/85
CPCC12N15/85C12M23/20C12M27/10C12M35/04C12M35/08C12N2510/00C12N5/0636C12N15/87C12N2525/00C12M41/48C12M45/05G16B20/00
Inventor MILTENYI, STEFANSCHEFFOLD, ALEXANDER
Owner MILTENYI BIOTEC
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