Method for manufacturing a patient specific cell population using a multifunctional self-contained apparatus and tubing set
The invention relates to a system, comprising: a) a sample processing unit, comprising an input port and an output port coupled to a rotating container having at least one sample chamber, the sample processing unit configured provide a first processing step to a sample or to rotate the container so as to apply a centrifugal force to a sample deposited in the chamber and separate at least a first component and a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, the cell separation unit comprising separation column holder, a pump and a plurality of valves configured to at least partially control fluid flow through a fluid circuitry and a separation column positioned in the holder, the separation column configured to separate labeled and unlabeled components of sample flowed through the column.
1 . A method of preparing a therapeutic cell population from a population of donor cells for administration to a patient in need thereof, the method comprising:
reversibly installing a single-use assembly on a computer controlled apparatus;
wherein the single-use assembly is closed and sterile, and is thereby configured to prepare said therapeutic cell population from a donor cell population in a manner such that the therapeutic cell population is suitable for administration to a human patient;
wherein the single-use assembly includes the following components:
(1) a sample processing unit that includes an input port operably connected to a sample chamber, wherein the sample chamber comprises ports to supply fresh media and gasses to support culture of a sample of cells in the chamber, and is configured to wash target cells and/or adjust sample volume as needed,
(2) a sample separation unit that includes a magnetic separation member, and
(3) fluid circuitry that interconnects the sample processing unit and the sample separation unit, so that cells can be caused to flow between the sample processing unit and the sample separation unit in either direction;
wherein the computer controlled apparatus is configured to operate the sample processing unit, the sample separation unit, and the fluid circuitry of the single-use assembly in a sequence of steps chosen by an operator;
programming or operating the computer-controlled apparatus to process a population of donor cells in the single-use assembly to produce the therapeutic cell population by applying a plurality of steps selected from the following steps (a) to (e):
(a) positive or negative selection of a defined population of target cells using the magnetic separation member;
(b) activation or differentiation of cells by culturing with one or more cytokines or growth factors;
(c) transduction or transfection of cells by culturing with genetic material;
(d) expansion of cells before or after any of steps (a) to (c) by culturing in fresh media; and
(e) formulation of the therapeutic cell population in an adjusted volume of fresh liquid.
2 . The method of claim 1 , wherein the magnetic separation member is configured so that magnetically responsive particles or microparticles coated with monoclonal antibody specific for cell surface antigens can be used to select chosen cell populations.
3 . The method of claim 1 , wherein the magnetic separation member comprises an electromagnet that can be operated to retain or release magnetically labeled target cells.
4 . The method of claim 1 , wherein the sample separation unit is configured for positive selection of target cells,
whereby target cells labeled with magnetic particles are retained in the magnetic separation member, while non-target cells that are not labeled with magnetic particles pass into a waste container.
5 . The method of claim 1 , wherein the sample separation unit is configured for negative selection of target cells,
whereby non-target cells labeled with magnetic particles are retained in the magnetic separation member and discarded, while target cells that are not labeled with magnetic particles are retained.
6 . The method of claim 1 , wherein the components of the single-use assembly are constructed and arranged so that target cells can be isolated from other cells in the donor cell population by multiple loops of positive and negative selection using the magnetic separation member.
7 . The method of claim 1 , wherein the sample chamber is configured so that a cell population may be cultured therein for a sufficient time and under suitable conditions whereby the cell population is expanded.
8 . The method of claim 1 , wherein the transduction or transfection in step (c) is done by electroporation.
9 . The method of claim 1 , wherein the sample chamber comprises a window or prism through which to monitor cells in the sample chamber.
10 . The method of claim 1 , wherein the sample processing unit is configured to remove red cells and/or platelets before processing the donor cell population using the sample separation unit.
11 . The method of claim 1 , wherein the sample processing unit is configured to separate different cell populations by gradient centrifugation.
12 . The method of claim 11 , wherein the computer controlled apparatus comprises a holder for the magnetic separation member, a pump operating on the fluid circuitry, and a plurality of valves that control flow through the fluid circuitry and the magnetic separation member of the single-use assembly.
13 . The method of claim 1 , wherein the computer controls the flow of fluids through the fluid circuitry and sample separation unit, controls magnetic field strength in the sample separation unit to provide for retention and release of magnetically labeled target cells or analyte, and directs final collection products into appropriate containers.
14 . The method of claim 1 , wherein the computer is programmed to operate the apparatus to prepare a genetically modified cell population from a donor cell population in the single-use assembly by performing the following steps:
(a) receive a sample of cells through the input port into the sample processing unit;
(b) prepare the sample of cells in the sample processing unit;
(c) transfer the prepared cells from the sample processing unit to the sample separation unit;
(d) separate the prepared cells into target cells and non-target cells in the sample separation unit using the magnetic separation member, allowing non-target cells to pass to a waste container,
(e) transfer the separated target cells back to the sample processing unit;
(f) combine and culture the target cells with genetic material in the sample chamber, supplying gas and fresh media as needed so as to genetically modify the cells;
(g) process the genetically modified cells by washing and adjusting volume; and thereafter
(h) deliver the genetically modified cells as a therapeutic cell population to a product collection container.
15 . The method of claim 1 , wherein cells in the population of donor cells are autologous or allogeneic to the patient.
16 . The method of claim 1 , wherein the therapeutic cell population has been enriched for cells that have one or more of the following characteristics:
CD34 positive,
CD133 positive,
CD14 positive, and/or
CD8 negative.
17 . The method of claim 1 , wherein the therapeutic cell population is either:
a population of stem cells or progenitor grafts from cord blood;
a dendritic cell vaccine;
activated natural killer cells;
peripheral blood mononuclear cells;
an expanded population of T helper cells, or a population of antigen-specific T cells.
18 . The method of claim 1 , wherein the therapeutic cell population obtained thereby is genetically modified.
19 . A method of therapy of a human patient in need thereof, comprising:
obtaining a therapeutic cell population from a population of donor cells that have been prepared according to the method of claim 1 , and
administering the therapeutic cell population to the patient.
20 . The method of claim 19 , wherein the therapy is in the context of one of the following:
stem cell or progenitor cell transplantation for tissue regeneration,
cancer treatment,
treatment of an infectious disease,
tissue regeneration,
inducing immunological tolerance for the purpose of organ transplantation, or
inducing immunological tolerance for therapeutic management of an autoimmune disease.