Improved methods of cell culture for adoptive cell therapy

A technology of cells and culture medium, applied in the field of cultured cells

Inactive Publication Date: 2012-10-31
WILSON WOLF MFG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Limit medium volume to growth surface area with a maximum ratio of 1ml/cm 2 to al...

Method used

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  • Improved methods of cell culture for adoptive cell therapy
  • Improved methods of cell culture for adoptive cell therapy
  • Improved methods of cell culture for adoptive cell therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Demonstration of limitations of conventional methods.

[0058] The data of this example demonstrates that when using a media volume of 2 ml per well (i.e., a media height of 1.0 cm and a ratio of media volume to surface area of ​​1 ml / cm 2 ) standard 24-well tissue culture plate (that is, each well has a surface area of ​​2 cm 2 ) Limiting factors in conventional culture methods for preparing EBV-CTL.

[0059] Stage 1 of culture, day 0:

[0060] By using antigen-presented γ-ray irradiated (40Gy) autologous EBV-LCL at a PBMC:LCL ratio of 40:1 and 1ml / cm 2 The ratio of medium volume to growth surface cell composition of PBMC from normal donors (approximately 1×10 6 cells / ml) were cultured to start the expansion of the EBV-CTL population, thus supplemented with 45% Click medium (Irvine Scientific, Santa Ana, CA), 2mM GlutaMAX-I, and 10% FBS (fetal bovine serum). RPMI 1640 medium to establish approximately 1 x 10 6 cells / cm 2 surface density of the ...

Embodiment 2

[0082] Example 2: At any given stage or at the beginning of each stage of culture, a reduction in the amount of time required to increase a target cell population can be achieved by reducing the cell surface density of the target cell population.

[0083] We hypothesized that the reduced expansion rate of the target cell population after the second T cell stimulation compared to the first stimulation is due to restrictive cell culture conditions leading to activation-induced cell death (AICD). For example, refer to image 3 A, At the first stimulation, the EBV antigen-specific T cell component of PBMCs corresponds to at most 2% of the cell population, so the seeding density of antigen-specific responsive T cells is less than 2 × 10 4 / cm 2 , the remaining PBMC act as non-proliferative feeder cells (eg image 3 CFSE-positive cells in A), these feeder cells maintain optimal cell-cell contacts to enable proliferation of antigen-specific CTLs. In contrast, at the second stimu...

Embodiment 3

[0087] Example 3: Minimum Surface Density of Cell Populations Comprising Target Cells and / or Antigen Presenting Cells can allow growth of target cell populations seeded at very low surface densities.

[0088] Figure 4 shows when continuing image 3 The work shown in is an example of the results we obtained, which further demonstrates that when the target cell needs the support of other cells, as long as the target cell is provided with sufficient feeder cells and / or antigen presenting cells, unconventional low Target cell surface density can initiate population expansion. In these experiments, we went on to demonstrate that at an R:S ratio of 8 to 1 approximately 1.0×10 6 Target cells / cm 2 Compared to only about 3900 target cells / cm at an R:S ratio of 1:32 2 We stopped testing at how well the total cell composition of the target cells greatly expanded beyond 50-fold the starting surface density.

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Abstract

An improved method of culturing cells for cell therapy applications that includes growing desired cells in the presence of antigen-presenting cells and/or feeder cells and with medium volume to surface area ratio of up to 1 ml/cm2 if the growth surface is not comprised of gas permeable material and up to 2 ml/cm2 if the growth surface is comprised of gas permeable material. The desired cells are at a surface density of less than 0.5x106 cells/cm2 at the onset of a production cycle, and the surface density of the desired cells plus the surface density of the antigen presenting cells and/or feeder cells are at least about 1.25 xlO5 cells/cm2.

Description

technical field [0001] The present invention relates generally to methods of culturing cells, and more particularly to the culturing of cells for cell therapy. Background technique [0002] Cell culture is a major contributor to the cost and complexity of cell therapy. With current methods, the process of culturing cells is time-consuming and costly. In general, in vitro culture processes performed in various stages are employed in order to produce a large number of cells. In the initial stages, the cells of interest are a relatively small population in a composition of cells placed in a cell culture device. At this stage, the cellular composition typically includes a source of target cells (eg, peripheral blood mononuclear cells), feeder cells that stimulate the growth of the target cells and / or present antigens. Culture devices and methods that leave the medium in which cells reside generally undisturbed are advantageous because the cells remain relatively undisturbed. ...

Claims

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Application Information

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IPC IPC(8): C12N5/078C12N5/02
CPCC12N2502/11C12N5/0636
Inventor 胡安·F·维拉克莱奥·M·鲁尼安·M·里恩约翰·R·威尔森
Owner WILSON WOLF MFG
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