Sustained release of stem cell factor in a double network hydrogel for ex vivo culture of cord blood-derived CD34+ cells

Abstract

Objectives: Stem cell factor (SCF) is considered as a commonly indispensable cytokine for proliferation of haematopoietic stem cells (HSCs), which is used in large dosages during ex vivo culture. The work presented here aimed to reduce the consumption of SCF by sustained release but still support cells proliferation and maintain the multipotency of HSCs.

Materials and methods: Stem cell factor was physically encapsulated within a hyaluronic acid/gelatin double network (HGDN) hydrogel to achieve a slow release rate. CD34⁺ cells were cultured within the SCF-loaded HGDN hydrogel for 14 days. The cell number, phenotype and functional capacity were investigated after culture.

Results: The HGDN hydrogels had desirable properties and encapsulated SCF kept being released for more than 6 days. SCF remained the native bioactivity, and the proliferation of HSCs within the SCF-loaded HGDN hydrogel was not affected, although the consumption of SCF was only a quarter in comparison with the conventional culture. Moreover, CD34⁺ cells harvested from the SCF-loaded HGDN hydrogels generated more multipotent colony-forming units (CFU-GEMM).

Conclusion: The data suggested that the SCF-loaded HGDN hydrogel could support ex vivo culture of HSCs, thus providing a cost-effective culture protocol for HSCs.

Figure 1

A schematic representation of the SCF‐loaded hyaluronic acid/gelatin double network (HGDN) hydrogel fabricated via a two‐step photo‐crosslinking process. SCF was encapsulated within the hyaluronic acid/gelatin double network (HGDN) hydrogel to achieve a sustained release and support proliferation of haematopoietic stem cells (HSCs)

Figure 2

Scanning electron microscope (SEM) images of the Methacrylated hyaluronic acid (HAMA) hydrogel (A), methacrylated gelatin (GelMA) hydrogel (B) and hyaluronic acid/gelatin double network (HGDN) hydrogel (C), respectively. The scale bar was 50 μm

Figure 3

A, Representative stress‐strain curves of hydrogels under uniaxial compression. B, Calculated compressive modulus of curves at initial 10% strain. C, Swelling ratio of hydrogels in phosphate buffer solution (PBS). D, Release profiles of SCF from hydrogels at each specified time interval

Figure 4

(A) Cell viability and (B) expansion folds of MNCs during the 14‐day incubation on methacrylated hyaluronic acid (HAMA) hydrogel, methacrylated gelatin (GelMA) hydrogel and hyaluronic acid/gelatin double network (HGDN) hydrogel. MNCs cultured on plates were served as the control

Figure 5

Numbers of TNCs resulted from CD34⁺ cells cultured in plates without SCF (Condition I) or supplemented with SCF (Condition II), hyaluronic acid/gelatin double network (HGDN) hydrogel without SCF (Condition III) and SCF‐loaded HGDN hydrogels (Condition IV) at regular intervals over a period of 14 days. (P < .05; n = 3)

Figure 6

A, Representative density plots of CD34⁺ and CD34⁺ CD38⁻ phenotype of harvest cells on day 7 and day 14. B, Percentages of CD34⁺ and CD34⁺ CD38⁻ phenotype of cells cultured under 4 culture conditions (Conditions I‐IV). Isotype controls were used to establish the positive and negative quadrants (P < .05; n = 3)

Figure 7

The number of (A) CD34⁺ cells and (B) CD34⁺ CD38⁻ cells cultured under 4 culture conditions (Conditions I‐IV) was calculated on days 7 and 14 (P < .05; n = 3)

Figure 8

The number of colony‐forming unit (CFUs) granulocytes‐macrophage colony‐forming unit (CFU‐GM), granulocyte‐erythroid‐macrophage‐megakaryocytes colony‐forming unit (CFU‐GEMM), erythroid burst‐forming unit (BFU‐E) and total CFU) resulting from 500 CD34⁺ cells re‐isolated from expanded cells under 4 conditions for 14 days (P < .05; n = 3)