Optimizing culture medium for CD34<+> hematopoietic cell expansion

摘要

The present invention provides a method of determining the optimal composition of a serum-free, eukaryotic cell culture medium supplement, using 2-level factorial design and the steepest ascent method. The invention further provides a method of making a serum-free eukaryotic cell culture medium supplement and the generated thereof. The invention further provides a method of making a serum-free, eukaryotic cell culture medium and the medium generated thereof. The invention further provides a kit containing the medium of the invention. The invention also provides a method of expanding CD34<+> hematopoietic cells and a composition comprising CD34<+> hematopoietic cells in a serum-free, eukaryotic cell culture medium of the invention.

主权项

1. A method for producing a serum-free and cytokine-containing cell culture medium for CD34+ hematopoietic cell expansion, said method comprising:
a) providing CD34+ hematopoietic cells in a first cell culture medium, wherein the first medium is a basal medium supplemented with serum; b) finding a optimal kind and concentration of a first cytokine combination for optimal hematopoietic cell growth in the first cell culture medium by using a 2-level factorial design, wherein the 2-level factorial design is an experimental design used to study cell growth in the first cell culture medium supplemented with cytokine(s) and each cytokine at two-levels, present or absent, wherein the cytokine(s) in the cytokine combination has a positive regression coefficient, β, which is obtained using the 2-level factorial design, and wherein the cytokine combination is selected from the group consisting of IL-3, IL-6, EPO, Flt-3 ligand (FL), SCF, G-CSF, GM-CSF, TPO, and IL-6-sR; c) finding a serum substitute combination for optimal hematopoietic cell growth first cell culture medium by using a 2-level factorial design, wherein the 2-level factorial design is an experimental design used to study cell growth in the first cell culture medium supplemented with serum substitute(s) and each serum substitute at two-levels, present or absent, wherein the serum substitute(s) in the serum substitute combination has a positive regression coefficient, β, which is obtained using the 2-level factorial design, and wherein the serum substitute combination is selected from the group consisting of Lipid-rich bovine serum albumin, BSA (bovine serum albumin), TF (transferrin), glutamine, HC (hydrocortisone), peptone, 2-ME (2-mercaptoethanol) and insulin; d) using a steepest ascent method with step changes in the concentration of the cytokine constituent or serum substitute constituent based on foregoing results of 2-level factorial design and evaluating the effects on cell growth, wherein the steepest ascent method is a statistical and mathematical method used to approach the vicinity of the optimal concentration for cell growth; e) adopting the changed concentration that gives the most favorable result for CD34+ hematopoietic cell growth; and f) producing a second cell culture medium by mixing or admixing a basal medium with the cytokine combination and serum substitute combination at a concentration adopted in step (e), wherein the second cell culture medium is a cytokine-containing and serum-free cell culture medium.