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Human ex vivo immune system

A technique of immune system, culture system, applied in the field of methods and components

Inactive Publication Date: 2003-06-11
UNIVERSITY OF ROCHESTER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, the presence of specific cytokines (IL-1 and IL-4) is required, which is likely to reverse lymphopoiesis
[0010] Currently, there is a lack of a stable one-stage human lymphopoiesis model capable of generating not only B-cells but also other cells present in the bone marrow for studying the complex cell-cell interactions during lymphopoiesis type

Method used

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  • Human ex vivo immune system
  • Human ex vivo immune system
  • Human ex vivo immune system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] General Materials and Methods

[0149] Flow Cytometry - Hemocytometer Analysis

[0150] Lymphocyte subtypes (helper and cytolytic T cells and B cells) and percentages of activated lymphocyte surface markers were quantified by flow-cytometry on an EPICS Profile Analyzer (Coulter, Miami, FL). Cell samples are incubated with fluorescently labeled antibodies and isotype controls. Antibodies used were anti-CD3 (pan T cells), anti-CD4 (helper T cells), anti-CD8 (cytolytic T cells), anti-TCRαβ (with αβ T cell receptor T-cells), anti-TCRγδ (T-cells with γδ T cell receptor), anti-CD45RA (naive T cells), anti-CD45RO (activated T cells), anti-CD19, anti - CD20, anti-CD21, and anti-CD10 (B cells) (10). Immunocytochemistry

[0151] Acetone-fixed cytospin slide preparations of nonadherent cells in culture were detected by monoclonal (anti-CD3, anti-CD19, anti-CD56, and anti-TdT) or polyclonal antibodies (anti-cytoplasmic μ, anti-surface IgG, and anti-surface IgM) labeling, ...

Embodiment 2

[0165] 3D human long-term bone marrow culture

[0166] An appropriate number of monocytes (4-6×10 6 The porous microcarrier portion transferred into the bioreactor per culture chamber) is incubated with the culture. The cultures were grown in a humid incubator (containing 5% CO 2 ) at 37°C. LTBMC culture medium (changed every day), by 70% (v / v) McCoy's 5A culture medium (Gibco), 1 * 10 -6M hydrocortisone (Sigma, St.Louis, MO), 50 u / ml penicillin (Sigma), 50 mg / ml streptomycin (Sigma), 0.2 mM L-glutamine (Gibco), 0.045% sodium bicarbonate ( Sigma), 1×MEM sodium pyruvate (Gibco), 1×MEM vitamin solution (Gibco), 0.4×MEM amino acid solution (Gibco), 12.5% ​​(v / v) heat-inactivated horse serum (Gibco), and 12.5% Composition of heat-inactivated FBS (Gibco). The culture medium is supplemented with recombinant human stem cell factor (rhSCF 50ng / ml) and lymphocyte-specific lymphokines, IL-2 (rhIL2, 1000U / ml) and IL-7 (rh IL-7, 2ng / ml) . The cultures were fed daily with non-supp...

Embodiment 3

[0169] Detection of B lymphocytes

[0170] Flow cytometric analysis of cell outflow from three-dimensional human bone marrow simulations in the absence of exogenous growth factors identified pro-B (pro-B cells) (CD10 + ), immature B (CD19 + ), and mature B-cells (CD20 + , CD21 + )The presence. 2.4% of cells at week 0 expressed the marker CD10, which represented the pro-B (primary B cell) cell population in fresh bone marrow ( figure 2 ). The pro-B (pro-B cell) cell population was maintained at the same level after one week of culture. But at week 2, the pro-B (proto-B cell) cell population declined dramatically and only recovered at week 4. This fluctuation may represent the regenerative process that occurs in the three-dimensional culture and heralds the active B cell lymphopoiesis present in the bioreactor. Moreover, CD10 + B-cell populations (pro-B (pro-B-cell) cells) fluctuate with immature (CD19) and mature (CD20 + and CD21 + ) The fluctuations of B cells are...

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Abstract

The present invention provides cultured immune system cells and methods of producing same. The method comprises culturing stromal cells and hemopoietic stem cells or in a chamber having a scaffolding covered or surrounded with culture medium, wherein the scaffolding allows for hemopoietic stem cells and stromal cells to have cell to cell contacts in three dimensions. The subject immune system cells are useful for screening drugs which inhibit or stimulate the immune system. The subject immune system cells are also useful in treating diseases of the immune system.

Description

[0001] This application was originally filed on November 17, 1999 as provisional application 60 / 166,026. [0002] This invention was made with US Government support under National Science Foundation Contract No. 60 / 166,026 and thus the US Government has certain rights in it. technical field [0003] The present invention relates to the field of cell culture, and in particular to methods and components related to cultured immune system cells. Background of the invention [0004] All hematopoietic cell lineages, including erythrocytes, granulocytes, macrophages, lymphocytes, and megakaryocytes, are derived from a small class of cells called pluripotent stem cells (PCSs). PCs have the ability to self-renew or become specialized stem cells capable of forming specific hematopoietic differentiated cell lineages. Specifically, PSCs form pluripotent cells of myeloid or lymphoid lineage. These pluripotent cells in turn form unipotent ...

Claims

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Application Information

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IPC IPC(8): G01N33/50A61K48/00A61P37/00C07K16/00C12M3/06C12N5/02C12N5/0789C12N15/09C12P21/08C12Q1/68G01N33/15G01N33/569
CPCC12N2501/125C12N2501/23C12N2501/39C12N5/0647A61P37/00
Inventor J·H·D·吴A·曼塔拉里斯
Owner UNIVERSITY OF ROCHESTER
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