1) Recombinant human transferrin (Optiferrin) and recombinant human albumin (Cellastim) improved the expansion of mononuclear cells and hematopoietic stem cells compared to animal-derived components. 2) The combination of Optiferrin and Cellastim roughly tripled the expansion of mononuclear cells and doubled the expansion of CD34+ stem cells compared to the best non-recombinant combination. 3) Expanded CD34+ stem cells grown with Optiferrin and Cellastim differentiated normally into various hematopoietic cell types, demonstrating their ability to support downstream applications.

1. Recombinant Transferrin and Albumin Improves Mononuclear and
Hematopoietic Stem Cell Expansion
Steve Pettit1, Paul Price2
1. InVitria, Cell Culture Development, Fort Collins, CO, info@invitria.com, 800-916-8311 2. D-Finitive Cell Technologies; currently at Cellomics, Thermo Fisher Scientific, Pittsburgh, PA
Growth and expansion of the mononuclear cell population cells/well. The combination of Optiferrin and Cellastim was the most robust combination and improved cell growth/
proliferation to 44 x 103 cells/well.
Abstract After thaw, isolated mononuclear cells were seeded at 9.5 x 103 viable cells/well (6 replicate wells) in
serum-free base medium supplemented with various combinations of albumins, transferrins or iron Table 2b. Relative effect of transferrins, chelate and albumins on the proliferation of CD34+ cells after 6 days of
Improving the expansion of mononuclear and stem cells is a major challenge in chelate compound. The number of viable cells was determined after 5 days of growth. growth (pHSA/iron chelate = 100).
regenerative medicine. The majority of current serum-free stem cell expansion media Hu holo-transferrin (mg/L) Optiferrin rTransferrin (mg/L) Iron
Table 1a. The effect of transferrins, chelate and albumins on the proliferation of mononuclear cells after 5 days of Albumins
contain animal-derived components in order to enhance performance. The advantages 5 12.5 5 12.5 Chelate
of using animal-free recombinant transferrin and albumin as media components were growth (viable cells x 103 /well). plasma HSA – 1000 mg/L 75 113 (a) 150 175 100
Hu holo-transferrin (mg/L) Optiferrin rTransferrin (mg/L) Iron Cellastim rHA – 1000 mg/L 100 250 175 275 (b) 200
determined for the expansion of mononuclear and CD34+ stem cells isolated from Albumins
5 12.5 25 50 5 12.5 25 50 Chelate Comparative controls
umbilical cord blood. plasma HSA – 1000 mg/L 16 18 12 14 18 18 12 2 16 No albumin or iron source 0
Iron is required for cell growth. Recombinant human transferrin (Optiferrin™, InVitria) plasma HSA – 2500 mg/L 8 8 6 4 2 10 6 4 16 Invitrogen StemPro 34 (control) 100
Cellastim rHA – 1000 mg/L 34 44 18 14 50 48 44 42 34 188
was compared to plasma-derived transferrin and an iron chelate as iron sources in an Cellastim rHA – 2500 mg/L 8 16 20 14 16 18 12 12 10
Sigma Stemline II (control)
animal-component free expansion medium. In addition, recombinant human albumin a) best animal-derived solution = 113 b) best recombinant solution = 275
a) Initial number of cells plated: 9.5 x 103/well
Table 2b shows the relative effect of the various albumins and iron carriers (transferrins or iron chelate). The results
(rHA)(Cellastim™, InVitria) was compared to plasma-derived human albumin. Optiferrin Comparative controls
indicate that the combination of Cellastim and Optiferrin delivered 2.5-fold the performance of the best non-
improved both mononuclear and CD34+ cell expansion compared to the other iron No albumin or iron source 0 recombinant combination.
sources. Cellastim dramatically outperformed plasma-derived albumin for the expansion Invitrogen StemPro 34 (control) 6
of both cell populations. The combination of Optiferrin and Cellastim roughly tripled the Sigma Stemline II (control) 16 Estimation of the growth-phase expansion of CD34+ cells
performance of the best performing non-recombinant combination. Expanded CD34+ Table 1a shows the absolute viable cell counts. The combination of plasma HSA and iron chelate compound The fold expansion of CD34+ cells during the growth phase (from day 2-7 of incubation) was
produced, at a maximum, 16 x 103 cells/well. Human holo-transferrin performed roughly equivalent to iron chelate. determined. The combination of Optiferrin and Cellastim outperformed other combinations and
cells were successfully differentiated into the desired hematopoietic cell lineages. Supplementation with Optiferrin rtransferrin improved growth to 18 x 103 cells/well. The combination of Optiferrin
resulted in 11.6-fold expansion of the CD34+ cell population. The performance was more than double
The results indicate that Optiferrin rTF and Cellastim rHA improve the performance of and Cellastim dramatically improved cell growth to 50 x 103 cells/well.
that of the best non-recombinant combination (at 5.1-fold expansion).
cell expansion media. These recombinant components enable the development of high- Table 1b. The relative effect of transferrins, chelate and albumins on the proliferation of mononuclear cells after 5 Table 3. Estimated fold expansion of CD34+ cells from incubation days 2-7 in various combinations of transferrins,
performance animal-free expansion media. days of growth ( plasma HSA/iron chelate = normalized to 100). chelate and albumins.
Hu holo-transferrin (mg/L) Optiferrin rTransferrin (mg/L) Iron Hu holo-transferrin (mg/L) Optiferrin rTransferrin (mg/L) Iron
Albumins
Introduction and experimental approach 5 12.5 25 50 5 12.5 25 50 Chelate Albumins
5 12.5 5 12.5 Chelate
Recombinant human albumin (Cellastim, InVitria) was evaluated as a replacement for plasma HSA – 1000 mg/L 100 113 (a) 75 88 113 113 75 13 100 plasma HSA – 1000 mg/L 3.4 5.1 (a) 7.1 8.0 4.7
plasma HSA – 2500 mg/L 50 50 38 25 13 63 38 25 100
human plasma albumin (plasma HSA) in the cell culture expansion of mononuclear Cellastim rHA – 1000 mg/L 213 275 113 88 313 (b) 300 275 263 213
Cellastim rHA – 1000 mg/L 4.3 10.5 7.8 11.6 (b) 9.1
cells (MNCs) and hematopoietic stem cells isolated from human cord blood. In addition, Comparative controls
Cellastim rHA – 2500 mg/L 50 100 125 88 100 113 75 75 63
Invitrogen StemPro 34 (control) 5.0
recombinant human transferrin (Optiferrin, InVitria) was evaluated as a replacement Comparative controls
Sigma Stemline II (control) 8.1
for either human transferrin or a ferrous sulfate chelate as an iron supply for MNCs and No albumin or iron source 0
a) Best non-recombinant solution = 5.1 b) Best recombinant solution = 11.6
hematopoietic stem cell expansion. Invitrogen StemPro 34 (control) 38
Sigma Stemline II (control) 100 Differentiation capabilities of CD34+ cells expanded with recombinant
Materials and Methods a) Best animal-derived soluion = 113 b) Best recombinant solution = 313
human albumin and transferrin
Mononuclear cells were isolated from human cord blood via Histopaque-1077 Table 1b shows the relative effect of the various albumins and iron carriers. The results indicate that the Base test medium supplemented with optimal concentrations of Cellastim and Optiferrin was
best combination of Cellastim and Optiferrin delivered roughly triple the performance of the best non-
centrifugation and stored as a frozen cell bank. CD34+ stem cells were isolated recombinant combination.
compared to the commercial medium Stemline II (Sigma) for its ability to support cell lineage
from the mononuclear cells by the EASYSEP™ human CD34+ selection kit (Stem Cell differentiation in a colony-forming unit assay. Colonies were observed from all cell types (BFU-E,
Technologies) and stored as a frozen cell bank. Growth and expansion of the CD34+ cell population CFU-E, CFU-M, CFU-GM, CFU-GEMM). BFU-E was the predominant cell type observed for both media.
After thaw, isolated CD34+ cells were seeded at 5.17 x103 cells/well in serum-free medium with No differences were seen in the frequency of cell types that had been expanded in either medium.
The expansion of MNCs and CD34+ cells was evaluated in an animal-free, serum-free,
various combinations of albumins, transferrins or iron chelate compound (8 replicate wells). The number
hematopoietic expansion medium (HPE-, D-Finitive technologies) which contained
of viable cells was determined after 7 days.
no albumin or iron carrier (transferrin or iron chelate). The base medium was further
supplemented with various combinations of albumins and iron carriers in addition Table 2a. Effect of transferrins, chelate, and albumins on the proliferation of CD34+ cells over 6 days of growth Conclusions
to the standard growth factor cocktail (D-Finitive Cocktail #1). After incubation and (viable cells x 103/well).
cell growth, expanded cells were counted by trypan blue exclusion to determine the Hu holo-transferrin (mg/L) Optiferrin rTransferrin (mg/L) Iron Recombinant human albumin and transferrin were evaluated for their ability to
Albumins
combination of albumin and iron carrier with the most robust effect on cell proliferation.
5 12.5 5 12.5 Chelate support the expansion of mononuclear and CD34+ stem cells grown in an animal-
plasma HSA – 1000 mg/L 12 18 24 28 16
free cell culture expansion medium. The combination of Cellastim rAlbumin and
Expanded CD34+ stem cells that had been grown in medium supplemented with Cellastim rHA – 1000 mg/L 16 40 28 44 32
a) Initial number of cells plated: 5.7 x 103/well Optiferrin rTransferrin outperformed the best non-recombinant solutions by 2-3
Optiferrin and Cellastim were examined by colony-forming unit (CFU) assays induced
Comparative controls fold and produced CD34+ cells with similar differentiation capability. These data
by 3-D extracellular methylcellulose matrix according to the procedure recommended
No albumin or iron source 0 indicate that Cellastim rAlbumin and Optiferrin rTransferrin are effective media
by Miltenyi Biotec. After 17 days, the expanded cells were examined for the presence of
BFU-E, CFU-E, CFU-M, CFU-GM and CFU-GEMM colonies. The relative abundance of the
Invitrogen StemPro 34 (control) 6 supplements that improve the growth and expansion of mononuclear cells and
Sigma Stemline II (control) 30
populations were noted and compared to the Sigma Stemline II commercial medium hematopoietic stem cells. These recombinant media components enable the
Table 2a shows the absolute viable cell counts. The combination of plasma HSA and iron chelate produced, at a
as a control. maximum, 16 x 103 cells/well. Substitution of human holo-transferrin for iron chelate modestly improved cell growth development of high-performance animal-free expansion media.
to 18 x 103 cells/well. Substitution of Optiferrin for human holo-transferrin improved cell growth further to 28 x 103