Theoretical and Experimental Investigation of Alginate Microtube Extrusion for Cell Culture Applications.

A novel cell culture technology, consisting of hollow alginate tubes, OD ~550 µm, ID ~450µm containing a cell suspension, provides stress-free conditions. Cells reach confluency in approximately ten days with cell densities of 0.5 - 1 billion cells per mL. Tubes are manufactured in a tri-axial needle extruder with three concentric flows. The cell suspension flows in the inner needle (N1), the alginate solution flows in the annulus between N1 and the second needle (N2) and a CaCl 2 solution is the sheath fluid between the second and third needle (N3). Beyond the tip of N2, the sheath solution is in contact with the alginate and Ca 2+ diffuses into the alginate solution and crosslinks it to form an alginate microtube around the core fluid. The cross-linked layer moves radially inwards like a front, starting at the sheath/annulus interface and ends at the annulus/core interface. A mathematical model is used to find the minimum length z C of direct contact between the CaCl 2 solution and the alginate solution to complete the cross-linking. Experimental results support the theoretical findings that stable tubes can only be manufactured if the contact length exceeds z C . Experiments also show that the extruder configuration N3>N2 is best for alginate tube manufacture.

Automated Expansion of Primary Human T Cells in Scalable and Cell-Friendly Hydrogel Microtubes for Adoptive Immunotherapy.

Adoptive immunotherapy is a highly effective strategy for treating many human cancers, such as melanoma, cervical cancer, lymphoma, and leukemia. Here, a novel cell culture technology is reported for expanding primary human T cells for adoptive immunotherapy. T cells are suspended and cultured in microscale alginate hydrogel tubes (AlgTubes) that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses and confine the cell mass less than 400 µm (in radial diameter) to ensure efficient mass transport, creating a cell-friendly microenvironment for growing T cells. This system is simple, scalable, highly efficient, defined, cost-effective, and compatible with current good manufacturing practices. Under optimized culture conditions, the AlgTubes enable culturing T cells with high cell viability, low DNA damage, high growth rate (≈320-fold expansion over 14 days), high purity (≈98% CD3+), and high yield (≈3.2 × 108 cells mL-1 hydrogel). All offer considerable advantages compared to current T cell culturing approaches. This new culture technology can significantly reduce the culture volume, time, and cost, while increasing the production.

Scalable Culturing of Primary Human Glioblastoma Tumor-Initiating Cells with a Cell-Friendly Culture System.

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 108 cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.