Scanning electron microscopy image of (A) colloidal crystals made from 110 μm polystyrene beads, and (B) ICC scaffolds from silicate gel. (C) ICC scaffolds after 5 days in culture with HS-5 bone marrow stromal cells. (D) 3D reconstructed poly (acrylamide) hydrogel ICC scaffold after 5 bilayers of FITC-albumin and PDDA coating. The diameter of the spherical cavities is 110 μm, interconnecting pores is ca. 15-25μm. (E) 3D schematics of ICC topology and cell contacts within. Floating HSCs enter into a pore through interconnected channels that have diameters 2-3 times larger than that of a single cell. Temporarily entrapped HSCs undergo intense contacts with stroma cells residing on the pore surface.

Evaluation of bone marrow construct, bone marrow derived cells, peripheral blood and spleen cells after 2 weeks implantation of hydrogel ICC scaffolds on the backs of eight SCID mice. The scaffolds were seeded with CFSE labeled cord blood derived CD34+ HSCs and cultured for 3 days before implantation. (A) A high degree of vascularization is seen in the regions near the site of the implanted ICC construct. (B-G) Confocal microscopy images of 7 μm frozen sections of the ICC-bone marrow construct. DAPI (blue) and CFSE (green). (B) Human MHC-Class I (red), 400X (green channel was not overlaid for clarity). HSC markers: (C) CD34 (red), insert 630X (D) CD150 (red), insert, 630X and (E) CD133 (red), (F) CD19 (red) (green channel not overlaid for clarity) and (G) IgM expression (red), magnification 630X. (H) Flow cytometry evaluation of cell phenotypes found in the bone marrow construct, peripheral blood and spleens of SCID mice receiving constructs. For all flow cytometry data 10,000 events were collected for each sample. Isotype control staining was less than 2% cells positive for all antibodies used.

(A-C) Confocal microscopy images of 7μm sections of hydrogel scaffolds supporting CD34+ HSCs from cord blood. DAPI nuclear stain is blue for all images. (A) Nuclear RAG-1 (red) expression and surface expression of IgM (green), day 7, 200X. Arrows point to RAG-1 positive nuclei. (B) Cell surface co-expression of CD19 (red) and IgM (green), day 14, 630X. (C) Co-expression of cell surface IgM (green) and IgD (red) day 28, 630X. (D-E) Flow cytometric evaluation of cell surface expression of immunoglobulin M or D from one representative experiment for (D) IgM and (E) IgD. 10,000 events were collected for all samples, isotype controls shown as green overlays. (F) The average expression of CD40, IgM, IgD and IgM+IgD co-expression for plate and ICC cultures using CD34+ from cord blood (6 independent experiments). (G) Comparison of IgM production for LPS stimulated plate and ICC cultures from cord blood (CB), peripheral blood (PB) or bone marrow (BM) CD34+ HSCs. (H) Confocal micrograph of 7 μm section of ICC culture stained for expression of IgG (red) and CD105 (stromal cell marker, green). (I) Flow cytometry data for IgG versus IgM expression from one representative experiment showing class switch for cord blood CD34+ HSC cells after exposure to heat killed-influenza virus. 10,000 events each, isotype control data are on the left plot. (J) Evaluation of specific influenza antibody production by HAI assay, neutralizing antibody assay titer, anti-HA IgG antibody ELISA, and anti-nuclear protein (NP) ELISA (5 experiments each). Results are arranged according to the lowest dilution of culture supernatant exhibiting inhibition in case of HAI and neutralizing antibody or ng/ml concentrations of corresponding proteins in case of anti-HA and NP ELISAs.

(A-E) Confocal microscopy images of 7 μm frozen sections of hydrogel ICC scaffolds. (A) Stromal cells cultured for 3 days were stained with a CD105 stromal cell marker for visualization of the developing stromal cell network (green), 200X. (B) CD34+ HSCs (red) were seeded onto the ICC scaffold and imaged after 1 day of stromal cell culture, 400X. (C) Examination of the ICC scaffold cultures on day 28 showing stromal cells CD105 (green) and CD34+ HSC (red), 630X. All cell nuclei were stained with DAPI (blue). (D) Sections of 28 day ICC/HSCs cultures stained for actin (red) and CD34 (green), 630X. (E) Same cultures stained for CD150 (red). DAPI nuclear stain (blue) was used. Flow cytometry data for donor matched 3D ICC scaffold (upper row) and 2D plate (lower row) cultures for cells isolated from (F,G) bone marrow, (H,I) cord blood, and (J,K) peripheral blood. 10,000 cytometry events were collected for all samples. Solid red histograms show CD34 levels with isotype controls for each sample shown as the green line histogram overlay. (L) Comparative evaluation of CD34+ cells on day 28. Significantly more CD34+ cells were seen in ICC cultures for bone marrow (BM) (P=0.01), cord blood (CB) (P=0.004), or peripheral blood (PB) (P=0.03) than for donor matched 2D plate culture in a total of 6 experiments. (M) Confocal images of 28 day 2D plate culture (M, top left and right, 400x) and 3D ICC cultured cells (M, bottom, 400x left, 630x right) stromal cell peripheral blood derived CD34+ cell seeded cultures. Plate and ICC cultures were stained for cell surface expression of CD34 (red) and DAPI (blue) nuclear stain. There are few mitotic figures seen in CD34+ cells in plate (top micrographs) compared to the ICC (bottom micrographs) cultures. Numerous mitotic figures indicate proliferation of CD34+ cells (white arrows) were always seen in 28 day ICC but not in plate cultures. (N) Flow cytometric analysis of CFSE levels in cord blood derived CD34+ HSCs for ICC scaffold (N, bottom) and donor matched 2D plate cultures (N, top). Solid red histograms show CFSE level of CD 34+ cells on the same day as ICC scaffolds were seeded. Solid green histograms show CFSE levels on day 7 of culture. 10,000 cytometry events were collected for all samples. (O) Comparison of 2D versus 3D cell cultures in ICC scaffolds by HSC proliferation analysis using CFSE loss for CD34+-derived from bone marrow (BM), cord blood (CB) or peripheral blood (PB) (averages for 5 experiments).