Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells.
The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover, we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells, with high cell recovery (>80%) and viability (>95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration. Overall, an integrated process allowed for a shorter process time, recovering 70% of viable hMSC (>95%), with no changes in terms of morphology, immunophenotype, proliferation capacity and multipotent differentiation potential.
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
Bárbara
B
Cunha
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
Tiago
T
Aguiar
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
Marta M
MM
Silva
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
Ricardo J S
RJ
Silva
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
Marcos F Q
MF
Sousa
Repligen Corporation, MA, USA.
Earl
E
Pineda
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
Cristina
C
Peixoto
iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte da Caparica, Portugal.
Manuel J T
MJ
Carrondo
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal. Electronic address: mserra@itqb.unl.pt.
Margarida
M
Serra
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal. Electronic address: marques@itqb.unl.pt.
Paula M
PM
Alves
D000641:Ammonia / Q000378:metabolism; D019149:Bioreactors; D002448:Cell Adhesion; D002452:Cell Count; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D005347:Fibroblasts / Q000166:cytology; D052816:Foreskin / Q000166:cytology; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D010477:Perfusion; D019098:Telomerase / Q000378:metabolism
D005973:Glutamine; D019344:Lactic Acid; D000641:Ammonia; D019098:Telomerase; D005947:Glucose
Alternating tangential flow; Cell therapy; Mesenchymal stem cells; Perfusion; Process integration; Tangential flow filtration