In

We have developed protocols whereby a total of 30-90 x 10(6) hMSCs with an average viability greater than 90% can be produced in a single multilevel Cell Factory from a relatively small (1-3 mL) bone marrow aspirate in 14-20 d. It is possible to generate as many as 5 x 10(8) multipotent stromal cells (MSCs) from a single sample, depending on the number of Cell Factories seeded from the initial isolated hMSCs. Briefly, mononuclear cells are collected from a bone marrow aspirate by density gradient centrifugation. The cells are cultured overnight and the adherent cells are allowed to attach to the flask. Nonadherent cells are removed and the culture expanded for 7-10 d with periodic feeding of the cells. The cells are then harvested and seeded at low density (60-100 cells/cm2) into Nunc Cell Factories. The cells are allowed to expand for an additional 7-10 d, and are then harvested.

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10–14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6×107 infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

Successful retroviral gene transfer into hematopoietic cells has been demonstrated in a number of small and large animal models and clinical trials. However, severe adverse events related to insertional muta-genesis in a recent clinical trial for X-linked severe combined immunodeficiency reinforced the need to develop novel retroviral vectors with improved biosafety. Improvements include the use of self-inactivating (SIN) vectors as well as improvements in vector design. This chapter describes the basic design of gamma-retroviral and lentiviral SIN vectors that utilize a split-packaging system and includes a description of the various cloning modules frequently used in the design of such vectors that impact biosafety, titer, and transgene expression. In addition, this chapter describes the methods used for high titer vector production using calcium phosphate transfection both at research scale and at large scale for clinical application using a closed system bioreactor.