Table 1.
Characteristics of the UCB units after EloHAES separation and cryopreservation.
Table 2.
Characteristics of the CD34 CliniMACS separation on thawed UCB units.
Figure 1.
Ex-vivo generation of CD56+ NK cells from cryopreserved CD34+ UCB cells.
CD34-enriched UCB cells were expanded for two weeks and subsequently differentiated into NK cells for four additional weeks. Cell cultures were weekly analyzed for cell numbers and phenotype using flow cytometry. (A) Fold expansion of total cells for each donor after initial seeding of enriched CD34+ UCB cells during 6 weeks of culture using static Vuelife™ cell culture bags. (B) CD56+ cell frequency for each donor during the 6 week culture period for static bag cultures. (C) Fold expansion of total cells for each donor after initial seeding of enriched CD34+ UCB cell population during 6 weeks of culture using single use bioreactors. (D) CD56+ cell frequency for each donor during the 6 week culture period for bioreactor cultures. (E) Mean total CD56+ NK cell expansion during 4 weeks of differentiation using static bag (n = 3) or bioreactor cultures (n = 4). Data are depicted as mean ± SD. The asterisk (*) represents a p-value of <0.05.
Table 3.
Overview of the quantity and quality of final UCB-NK products generated from enriched CD34+ cells using static bags and single use bioreactors.
Figure 2.
Functional activity of ex vivo bioreactor-expanded NK cells before and after the washing process.
(A) Cytotoxicity of ex vivo-generated NK cells against K562 cells was analyzed after 18 hours of co-culture with unwashed (black bars) and washed (white bars) NK cells from three different donors at an E:T ratio of 1:1 or 10:1. (B) Degranulation of ex vivo-generated NK cells against K562 was analyzed by CD107a expression after 18 hours of co-culture after of unwashed (black bars) and washed (white bars) NK cells from three different donors at an E:T ratio of 1:1.
Figure 3.
Flow cytometry analysis of ex vivo bioreactor-expanded NK cells before and after washing.
The CD56+CD3− lymphocytes were analyzed of unwashed (A) and washed (B) NK cell products. A representative example out of three different NK cell products is shown.
Table 4.
Product release testing criteria and results of the final NK cell products.
Figure 4.
Stability tests of ex-vivo generated and processed NK cell products.
(A) The NK cell content of the processed final product was followed over time, while the products were either stored at 4°C or room temperature (RT) for a maximum of 3 days. The percentage of the CD45+/CD56+ cells is displayed from 3 different stability tests. (B) Viability of the final NK cell product was followed over time, while the products were either stored at 4°C or room temperature (RT). The percentage of the CD45+/CD56+/7-AAD− cells is displayed from 3 different stability tests.