key: cord-0270895-6l3qmugg authors: Loebel, Claudia; Weiner, Aaron I.; Katzen, Jeremy B.; Morley, Michael P.; Bala, Vikram; Cardenas-Diaz, Fabian L.; Davidson, Matthew D.; Shiraishi, Kazushige; Basil, Maria C.; Ochs, Matthias; Beers, Michael F.; Morrisey, Edward E.; Vaughan, Andrew E.; Burdick, Jason A. title: Microstructured hydrogels to guide self-assembly and function of lung alveolospheres date: 2021-09-01 journal: bioRxiv DOI: 10.1101/2021.08.30.457534 sha: 288d74894bf3b3bf438168fc5e49014644c8229a doc_id: 270895 cord_uid: 6l3qmugg Epithelial cell organoids have increased opportunities to probe questions on tissue development and disease in vitro and for therapeutic cell transplantation. Despite their potential, current protocols to grow these organoids almost exclusively depend on culture within three-dimensional (3D) Matrigel, which limits defined culture conditions, introduces animal components, and results in heterogenous organoids (i.e., shape, size, composition). Here, we describe a method that relies on polymeric hydrogel substrates for the generation and expansion of lung alveolar organoids (alveolospheres). Using synthetic hydrogels with defined chemical and physical properties, human induced pluripotent stem cell (iPSC)-derived alveolar type 2 cells (iAT2s) self-assemble into alveolospheres and propagate in Matrigel-free conditions. By engineering pre-defined microcavities within these hydrogels, the heterogeneity of alveolosphere size and structure was reduced when compared to 3D culture while maintaining alveolar type 2 cell fate of human iAT2 and primary mouse tissue-derived progenitor cells. This hydrogel system is a facile and accessible culture system for the culture of primary and iPSC-derived lung progenitors and the method could be expanded to the culture of other epithelial progenitor and stem cell aggregates. 2 human iAT2 and primary mouse tissue-derived progenitor cells. This hydrogel system is a facile and accessible culture system for the culture of primary and iPSC-derived lung progenitors and the method could be expanded to the culture of other epithelial progenitor and stem cell aggregates. Organoids have received considerable attention for modeling organogenesis and disease, facilitating large screening of therapeutic molecules (e.g., proteins, drugs), and for the sourcing of cells for therapeutic transplantation 1, 2 . With regards to the lung, organoids have primarily been applied to address questions in pulmonary biology, such as reparative mechanisms of lung progenitors and their responsiveness to regenerative and therapeutic molecules 3 . Given the importance of gas-exchange as the most fundamental function of the lungs, alveolar organoids (i.e., alveolospheres) are becoming an indispensable tool for in vitro studies, including for the modeling of distal lung injuries such as SARS-CoV-2 infections 4-6 . The alveolar epithelium comprises two distinct epithelial cell types: Type 2 cells (AT2), which are surfactant -producing alveolar progenitor cells that can self-renew and differentiate into type 1 cells (AT1) that cover the majority of the surface of the lung alveoli 7, 8 . When cultured in Matrigel and with mesenchymal feeder cells, primary mouse-derived AT2 cells form alveolospheres that comprise both AT1 and AT2 cells but often do not reproduce the structure of alveoli in adult lungs 3 . Importantly, recent advances in the differentiation of induced pluripotent stem cells (iPSCs) into AT2 cells have also enabled the culture of human alveolospheres, overcoming some of the challenges in the isolation and culture of primary human AT2 cells 9 . Although the spontaneous assembly and propagation of organoids within Matrigel have resulted in significant advances, artificial niches are increasingly being developed to provide distinct chemical and physical signals to organoids during culture 10, 11 . Specifically, synthetic niches can guide cellular selfassembly while overcoming challenges in Matrigel organoid cultures, such as the inherent variability in organoid formation efficiencies and morphology, which poses issues for standardizing organoid models 4 To illustrate the potential of hydrogels to enable assembly of alveolosphere organoids, we first employed human iPSC-derived alveolar progenitor cells (iAT2s) with a GFP knock-in cassette in one allele of the SFTPC gene, a specific marker for AT2 cells (Fig. 1A) 24 . Using a recently developed protocol 9,25 , we differentiated iPSCs (RUES2 line) into foregut endoderm followed by sorting for NKX2.1+ putative lung primordial lung progenitors and consecutive enrichment for SFTPC GFP+ cells (Fig. 1A) . The differentiation protocol resulted in a total yield of ~55% SFTPC GFP+ cells on day 66 in Matrigel (Fig. 1B) . The resulting SFTPC GFP+ cells were further purified and passaged as alveolospheres in the presence of lung maturation additives and selective Rho-associated kinase (ROCK) inhibitor, Y-27632 from day 0-2. CHIR was added back following 7 days of withdrawal (days 2-9) to increase efficiency of iAT2 maturation 9 (Fig. 1C) . Thus, this protocol allows for iAT2 serial passaging upon dissociation into single cells and formation of alveolospheres that retain SFTPC GFP+ expression in Matrigel. To demonstrate alveolosphere formation in synthetic hydrogels, iAT2s were embedded within hyaluronic acid (HA) hydrogels (norbornene-modified HA, modified with cell-adhesive peptide (HA RGD), 5% Matrigel (HA 5% MA) or 2 mg/mL Laminin/Entactin (HA Lm), all with a storage modulus of ~500 Pa (Fig. 1D ). At 14 days, small alveolospheres were observed in the HA RGD hydrogel, whereas culture in HA MA and HA Lm hydrogels resulted in increased formation of alveolospheres; however, the area was lower than MA controls ( Fig. 1E ). Alveolosphere formation further depended on the concentration of Matrigel and Laminin/Entactin, initial storage modulus and iAT2 seeding density ( Supplementary Fig. 1 ), indicating that the formation of alveolospheres is influenced by both chemical and mechanical signals. Alveolospheres formed within HA Lm hydrogels and expressed SFTPC GFP+ (Fig. 1F) , with a colony forming efficiency that was similar to Matrigel ( Supplementary Fig. 2) . Furthermore, quantification of SFTPC GFP+ expression confirmed that alveolospheres maintain their AT2 progeny with an average of ~31% SFTPC GFP+ cells (Fig. 1G) . Given that HA Lm hydrogels support alveolosphere growth, we next assessed whether iAT2s retain proliferative potential during serial passaging, consistent with previous protocols in Matrigel 25 , but using a modified enzymatic digestion (1 mg/mL hyaluronidase, no dispase). Using hyaluronidase digestion and 5 subsequent trypsin digestion into single cell suspensions, over a period of 3 passages, iAT2s reformed spheres ( Supplementary Fig. 3 ) with stable efficiency and proliferation kinetics (Fig. 1H) . These findings indicate that iAT2-derived alveolospheres can be formed and maintained in laminin/entactin-enriched synthetic hydrogels, enabling culture in a well-defined Matrigel-free 3D environment. However, this strategy presents relatively high heterogeneity, because alveolospheres form randomly, similar to traditional Matrigel culture. Thus, although well-defined HA hydrogels improve culture conditions, limited control over size and shape and difficulties of standardizing towards downstream analysis remain to be addressed. 3D hydrogels as a single cell suspension with 500 cells per µL and cultured with CHIR withdrawal between day 3-7 8 and CHIR addback (days [9] [10] [11] [12] [13] [14] . D Representative images of alveolospheres formed in Matrigel and hyaluronic acid hydrogels at 14 days. Hyaluronic acid hydrogels were crosslinked with a protease sensitive crosslinker via visible light-initiated thiol-ene reaction either modified with a cell-adhesive peptide (1 mM RGD), supplemented with 5% (wt/vol) Matrigel or 2 mg/mL laminin/entactin and with crosslinker amount adjusted to achieve an initial storage (elastic) modulus of 500 Pa (scale bar 100 µm, see supplementary figure 1 for representative images and quantification of viability and projected alveolosphere area in hyaluronic acid hydrogels supplemented with various concentrations of Matrigel, laminin/entactin and the influence of elastic modulus and cell seeding density). E Quantification of projected alveolosphere area at 14 days measured from brightfield images (*p<0.05, ****p<0.0001 by ANOVA and Bonferroni's multiple comparisons test, n = 155 (Matrigel), n = 55 (HA RGD), n = 106 (HA Ma), and n = 154 (HA Lm)). F Representative images of SFTPC GFP+ alveolospheres in HA LM hydrogels at 14 days (cell mask membrane stain (magenta), nuclei (grey), and SFTPC GFP+ expression (green), scale bar 100 µm). G Quantification of SFTPC GFP+ per alveolosphere area (cell membrane stain) at 14 days (ns = not significantly different by unpaired two-tailed t-test). H Quantification of the number of alveolospheres and proliferation kinetics of cell yield per re-embedded SFTPC GFP+ cells in HA Lm hydrogels over three passages. Given that iAT2s were able to form alveolospheres within a Matrigel-free hydrogel, we next sought to further control the growth and homogeneity of formed alveolospheres, a critical requirement for downstream readouts. Thus, we engineered hydrogel substrates that comprise microwell-shaped cavities for aggregation and individual alveolosphere formation through geometrical constraints (Fig. 2) . Accessibility and customization of such engineered systems is often the bottleneck for broader applications within the regenerative biology community. Here, we used commercially available cell culture surfaces (EZSPHERE TM ) with evenly spaced microwells to make silicone replica molds of a desired size and shape ( Supplementary Fig. 4 ). HA hydrogels were then fabricated with the silicone molds upon ultraviolet light mediated photo-curing onto a glass coverslip ( Fig. 2A , i) and subsequent transfer of the fabricated microwell hydrogels directly onto conventional tissue culture dishes ( Fig. 2A, ii) . Using this fabrication technique, hydrogels can be readily fabricated and are compatible with a range of different sizes as shown by fluorescent images of individual microwells (Fig. 2B ). As the microwell size can be accurately modulated, we fabricated hydrogels (~20 kPa) of small, medium and large microwell sizes, ranging from an average of 80-300 µm in depth (amplitude) and 250-810 µm in width ( Fig. 2C ). Hydrogel structures were stable during 14 days in culture in alveolosphere media with minimal changes in amplitude and width of individual microwells ( Supplementary Fig. 5 ). To test whether the hydrogel microwells support alveolosphere formation, we seeded iAT2s with an average density of 2 cells/µm 2 hydrogel surface area in alveolosphere media. In silico modeling predicted cell sedimentation into individual microwells and brightfield imaging confirmed the formation of differently sized and irregularly shaped aggregates within 1 hour upon seeding (Fig. 2D ). Formation of alveolospheres was controlled by the microwell size and initial seeding density, as measured by the projected alveolosphere area and viability. Within 14 days, iAT2 aggregates gave rise to alveolospheres across all microwell hydrogels ( Supplementary Fig. 6 ) and alveolosphere area depended on microwell size (Fig. 2E ). Although the efficiency and area increased in large microwells, alveolospheres also showed significantly reduced cell viability (~70%, Having shown that alveolospheres form in microwells, we used medium-size patterns to assess how iAT2 seeding density regulates growth and AT2 progeny within these engineered environments. Recent studies in intestinal organoids suggest a minimal number of cells is required to generate epithelial structures 21 . In addition, the initial aggregate size may induce paracrine signaling that directs self-assembly and growth of alveolospheres 21 . Thus, we seeded iAT2s at an average density of 15, 75 and 750 cells per individual microwell and monitored alveolospheres growth. At day 14, alveolospheres formed through all conditions with polarized apical surfaces (Fig. 3A) , which is consistent with the epithelial structures observed in 3D hydrogels ( Fig. 1) . During the process of iAT2 self-assembly, alveolospheres grew in size as indicated by the increasing alveolosphere areas with culture with starting populations of 75 cells per well and higher, whereas lower seeding densities failed to generate growing alveolospheres (i.e., 75 cells, Fig. 3B ). Similar growth patterns were observed in smaller microwells, further supporting that alveolosphere assembly and growth depends on initial iAT2 seeding densities ( Supplementary Fig. 8 ). The ability of iAT2s to generate alveolospheres is driven by their self-renewal potential and capacity to proliferate 9 , which may be influenced by the cell seeding density. EdU incorporation was used to visualize proliferating cells during the initial 7 days of culture (Fig. 3C ). While we observed proliferating iAT2s throughout all conditions, the percentage of EdU+ cells was increased for alveolospheres formed from higher cell seeding densities (Fig. 3D ). The increase in proliferative capacity further resulted in larger diameter alveolospheres at day 7 (Fig. 3E) , suggesting the influence of cell-cell contact and paracrine signaling. In addition, quantification of alveolosphere forming efficiency revealed that individual microwells retained 36±14% of the alveolospheres with low cell seeding densities (15 cells), whereas greater efficiencies of 73±21-98±3% were maintained in microwells at higher seeding densities of 75 and 750 cells (Fig. 3F ). We next sought to determine whether the microwell culture method supported AT2 progeny of alveolospheres, focusing on the fluorescent reporter and expression of surfactant protein C (pro-SFTPC), which is highly specific to AT2 cells 26 . At day 14, we observed SFTPC GFP+ expressing cells in all alveolospheres that were also expressing pro-SFTPC, as identified by immunostaining (Fig. 3G) . Varying the initial cell seeding density had little influence on SFTPC GFP+ levels, which ranged from 20-30%; however, lower cell seeding densities increased pro-SFTPC expression (Fig. 3H ). This is consistent with a previous study that altered iAT2 plating densities to enhance AT2 progeny within Matrigel 25 . We next sought to define the cellular heterogeneity of alveolospheres and their AT2 progeny using single-cell RNA sequencing (scRNA-seq) and protein identification. To assess the functionality of microwell-cultured alveolospheres we used one condition (75 cells) in comparison to Matrigel cultured alveolospheres. At 14 days, cells clustered into 7 different clusters as visualized by Uniform Manifold Approximation and Projection (UMAP) and we identified several AT2 specific markers expressed by cells cultured under both conditions, including SFTPC and SFTPB (Fig. 4A, Supplementary Fig. 9 ). This data suggests that cells within alveolospheres similarly represent AT2-like cells. Interestingly, a greater proportion of the total cells in microwell conditions maintained expression of mature AT2 markers, suggesting microwell hydrogels may provide some advantages over Matrigel in terms of maintaining AT2 fate in alveolospheres. In addition, previous studies showed that iAT2 cells in Matrigel exhibit some aspects of AT2 cell maturation including apical tight junctions, apical microvilli, and the expression of lamellar-like bodies 9 (Fig. 4B) . Therefore, we next assessed whether cells were able to process pro-SFTPs to mature SFTPB and SFTPC proteins that are exclusive to AT2 cells and essential components of surfactant 26, 27 . As a comparison, proteins were also extracted from human primary AT2 cells, alveolospheres cultured in Matrigel, and undifferentiated iPSCs. Western blots immunostained with antibodies that recognize the fully mature 8-kDa form of SFTPB and SFTPC revealed production of mature forms of each protein. Primary human AT2 controls and cells from both Matrigel and microwell-cultured alveolospheres also expressed the mature 8-kDa SFTPB and SFTPC proteins, whereas no staining of undifferentiated iPSCs was detected (Fig. 4C ). As such, the microwellcultured alveolospheres seem to efficiently process surfactant proteins into their mature form. Next, we assessed whether the iAT2s in alveolospheres also formed lamellar bodies, the functional organelles in which surfactant is stored before exocytosis into the air spaces to form a phospholipid-rich film at the airliquid interface 27 . Using transmission electron microcopy (TEM) analysis of primary human AT2 cells, these organelles are characterized by a tight packing of lipid lamellae into lamellar bodies. Similarly, Matrigel and microwell-cultured alveolospheres revealed lamellar-body like inclusions with a subset of inclusions expressing dense cores indicating the ongoing process of maturation (Fig. 4D) . Importantly, microwell-cultured alveolospheres further showed typical characteristics of epithelial differentiation, including the formation of tight junctions at the apical part of the lateral cell membrane and microvilli on the apical cell membrane (Fig. 4D ). These results suggest that alveolospheres when cultured in microwell hydrogels contain cells that form functional lamellar body-like inclusions, consistent with Matrigel cultures and findings reported in mature AT2 cells in vivo 26, 28, 29 . Having shown that microwell-cultured alveolospheres retain adult AT2 phenotypic markers in vitro, we next sought to assess their viability and differentiation capacity in vivo. Recent studies have shown orthotopic transplantation of lung epithelial cells into injured lungs as a functional tool to interrogate the responsiveness of cells to in vivo signaling cues [30] [31] [32] [33] [34] . Here, we used bleomycin induced tissue injury, characterized by spatial AT2 cell loss within damaged regions 35 (Supplementary Fig. 10 ). Immunodeficient non-obese diabetic (NOD) severe combined immunodeficiency (SCID) gamma (NSG) mice were injured with bleomycin at day 0, followed at day 10 by intranasal inhalation of alveolar progenitors derived from microwell and Matrigel-derived alveolospheres upon digestion (Fig. 5A ). Immunostaining of human-specific nuclear and mitochondrial markers at day 14 post-transplantation showed discrete clusters of human cells, predominantly at the border of damaged alveolar regions in each of the recipient mice (n = 3 per group). Human cell clusters appeared to adopt similarity to neighboring alveoli and retain at least some alveolar differentiation, as indicated by some cells being positive for pro-SFTPC (Fig. 5B) . Furthermore, staining for Ki67 expression revealed several proliferating cells that were located across the human cell clusters ( (Fig. 5C ). When analyzing cell clusters and percentage of Ki67+, we observed minimal differences between cells transplanted from alveolospheres cultured in Matrigel and microwells ( Supplementary Fig. 11 ). The lack of Keratin 5 expression in both conditions further confirmed that transplanted cells did not trans-differentiate into basal cells ( Supplementary Fig. 12 ). Although in contrast to recent findings on the differentiation of transplanted primary AT2 cells into basal cells 32 , the ability of cells to respond to the in vivo niche may depend on their origin (e.g., primary vs iPSC-derived), further highlighting the potential of the orthotopic transplantation assay to probe cell plasticity. We did not detect RAGE (receptor for advanced glycation endproducts), suggesting minimal differentiation into type 1 alveolar epithelial cells. Explanations for the lack of differentiation could be that the in vitro culture conditions are not supportive of AT1 differentiation, 9 the early time points upon transplantation, or the incompatibility between murine and human growth factors / ECM components that are required for efficient AT1 fate adoption. These findings suggest that alveolospheres when cultured within engineered microwell hydrogels can retain their proliferative capacity upon transplantation into injured murine lungs, consistent with results in Matrigel and previous observations 36 . However, the lack of AT1 cells in both culture conditions as well as transcriptomic differences when compared to primary AT2 cells 9 indicate that further optimization is still required for future applications, including cell-based therapies and disease modeling studies. While iPSC-derived alveolospheres have evolved as a reliable source of human AT2 cells, generation of primary alveolospheres continues to be critical, including for studies on mesenchymal to epithelial crosstalk and AT2 to AT1 differentiation 3,7,37 , which are currently not addressable in human alveolospheres 9 . Similarly, existing primary alveolosphere culture models depend almost exclusively on Matrigel as a 3D matrix, limiting their applicability for many downstream applications 12, 38 . Thus, to further validate our microwell hydrogel system, we next adapted our design for the generation and culture of primary murine alveolospheres. Commonly, co-culture with mesenchymal cells is necessary as stromal support for alveolosphere development 7 . To address this, hydrogel microwells were fabricated with murine lung fibroblasts that are embedded during crosslinking, enabling mesenchymal to epithelial cell crosstalk during culture (Fig. 5C ). Fibroblast cells were encapsulated at a density of 5 x10 6 mL -1 and we confirmed that embedded cells did not alter microwell fidelity (Supplementary Fig. 13 ). To generate alveolospheres, SFTPC EYFP+ AT2 cells were isolated from SFTPC CreERT2 R26R EYFP adult mice (Supplementary Fig. 14) and seeded directly into fibroblastladen hydrogel microwells (75 cells per well), following our established protocol. Within 7 days, isolated AT2 cells assembled into lumenized structures that were physically separated from the embedded fibroblasts while maintaining high cell viability (Fig. 5D ). Alveolospheres formed in the majority of individual microwells (~75%, Fig. 5E ), consistent with the efficiency of iAT2-derived alveolospheres (Fig. 3F,G) ; however, Sftpc EYFP+ expression levels were slightly lower when compared to Matrigel controls (Fig. 5F ). We also observed that direct co-culture through the mixing of fibroblasts and SFTPC EYFP+ AT2s resulted in similar alveolospheres, whereas no alveolospheres were found in the absence of fibroblasts ( Supplementary Fig. 15 ), supporting the importance of mesenchymal signaling during the formation of primary alveolospheres. These findings suggest that microwell hydrogels can be used for the generation of primary alveolospheres in a Matrigel-free environment, critical for a range of applications such as in modeling disease and probing mesenchymal to epithelial cell crosstalk. We have designed a versatile Matrigel-free culture system to generate lung alveolospheres either when embedded within 3D hydrogels or seeded atop microwell hydrogels. Previous studies to engineer organoids within microwell hydrogels have focused on the development of intestinal, pancreatic and cancer organoids 21- prevents their translation across groups. By using commercially available culture dishes with pre-formed microwells, we fabricated cytocompatible hydrogels with evenly spaced microcavities, which enabled the generation and culture of functional alveolospheres. Our data indicate that within microstructured hydrogels, human alveolospheres maintain their proliferative and differentiation capacity. This approach further supports the versatility of embedding other cell types, such as lung fibroblasts, into the hydrogel to enable the formation of primary mouse alveolospheres. The supportive mesenchyme was found to provide paracrine signals that are essential for alveolar epithelial cell self-assembly and organization. Thus, the functional encapsulation of fibroblast populations in engineered, functional alveolosphere cultures provides new perspectives on disease modeling. We anticipate that by modulating specific culture conditions such as fibroblast lineages, hydrogel stiffness and composition, the microwell culture system can be used to study the role of mesenchymal-toepithelial cell communication 37 and biophysical signaling during in vitro alveologenesis. AT2 functionality was tested through analysis of surfactant protein processing and survival upon orthotopic transplantation of dissociated alveolospheres into lungs of immunocompromised mice. When microwellcultured alveolospheres were transplanted into injured murine lungs, dissociated cells adopted alveoli-like structures while maintaining their proliferative capacity, and this was similar to alveolospheres formed within Matrigel. Previous work has reported data on the capacity of orthotopic transplanted epithelial progenitor cells to survive in injured murine lungs 31, 32, 34 . However, the technology described here could hold promise as a means to expand functional epithelial cells in Matrigel-free conditions for therapeutic applications. Further studies are required to probe whether cells are structurally and functionally integrated into mouse lungs, such as clonality and long-term replacement of injured epithelium; yet, to date, little is known regarding methods that confirm functional engraftment, or even the definition of engraftment in this context 33 . Although orthotopic transplantation assays are not yet sufficient to assess the contribution of transplanted cells to organ function, it provides a powerful platform to probe iPSC-derived lung epithelial cell viability, plasticity, and ability to incorporate into a compatible host tissue. Finally, although we observed lamellar inclusions and the functional capacity to process SFTPB and SFTPC to their mature form, extracted proteins from both conditions present relatively lower amounts when compared to primary AT2 cells. While these differences might, in part, be explained by the effect of in vitro culture and sample processing, issues of limited characterization remain to be eliminated, further highlighting the need for culture platforms that support culture of both iPSC-based and primary AT2 cells. Taken together, there are currently no strategies for the defined culture of lung alveolospheres in Matrigelfree conditions. Thus, the microwell hydrogels described herein provide means as an accessible culture system for the generation and maintenance of primary and iPSC-derived lung progenitors, which may be extendable to other epithelial progenitor and stem cell aggregates. The R26R EYFP+ mouse line was purchased from Jackson Laboratories and the Sftpc CreERT2 mouse line generously provided by Dr. Hal Chapman, and genotyping information has been previously described 39 . All mice were maintained on a mixed background and were 3-5 weeks of age for experiments in this study. (CK+DCI medium), containing 10 µM Y-27632 for the first 48 h after plating, followed by 5 days in K+DCI (without CHIR99021) and 7 days in CK+DCI. Alveolosphere were passaged every 14 days by digesting the Matrigel with 2 mg/mL dispase for 1h at 37°C, followed by incubation in 0.25% Trypsin/EDTA for 10 min at 37°C to obtain a single cell suspension. Cell quantification and viability were assessed using Trypan blue. Finally, cells were mixed with Matrigel, 50 µL drops formed within 24 well plates and incubated for 30 min at 37°C and 5% CO 2 . Electron Microscopy: Alveolospheres were released from Matrigel using dispase or mechanically retrieved from microwells through several PBS washes, followed by fixation in 2.5% glutaraldehyde in 0.1% cacodylate buffer for at least 3 h at room temperature. Sample preparation was performed as recently reported 9 . Briefly, dehydration was performed with acetone on ice and graded ethanol series. Samples were then incubated in 100% acetone at RT for 2x10 min and in propylene oxide at RT for 2x15 min. Finally, samples were embedded in Embed-812 (Electron Microscopy Sciences), incubated in uranyl acetate and lead citrate and imaged with a JEOL 1010 electron microscope including a Hamamatsu digital camera (AMT Advantage image capture software). Western Blotting: Western blots were performed to detect processed SFTPC and SFTPB protein as previously described 41 . Briefly, total protein content of cell lysates was assayed by the Bradford method followed by SDS-PAGE and immunoblotting. Western blotting used a previously published polyclonal pro-SFTPB antiserum ("PT3-SP-B" at 1:3000 dilution), a commercially available mature SFTPC antibody (WRAB-76694; Seven Hills Bioreagents at 1:2500 dilutiion), and Beta-Actin (Sigma Aldrich A1978 at 1:10000 dilution) followed by an HRP-conjugated secondary antibody and visualization by enhanced chemiluminescence. Hydrogel preparation and seeding: Hydrogel synthesis: Norbornene-modified Hyaluronic acid (NorHA) was synthesized as described previously 42 . The degree of modification was 26% by 1 H NMR (Supplementary Fig. 15 ). Enzymatically (metalloproteinase (MMP)) degradable di-thiolated peptides (GCNSVPMSMRGGSNCG) and thiolated cell-adhesive RGD peptides (GCGYGRGDSPG) were purchased from Genscript. NorHA hydrogels were fabricated by thiol-ene addition crosslinking with either ultraviolet (microwells) or visible light (3D hydrogels) and the photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Colorado Photopolymer Solutions). iAT2 encapsulation and culture: Hydrogel precursor solutions (4wt% polymer) were mixed with 1000 cells per µL (or as otherwise noted), 1 mM thiolated RGD and mixed with or without laminin/entactin (Corning, 354259) or Matrigel at different concentrations, and photo-polymerized with MMP-degradable peptide crosslinkers (400-500 nm, Omnicure S1500, Exfo) for 10 min at 10 mW cm -2 . Gels were crosslinked as 50 µL droplets atop thiolated coverslips and cultured in 48-well plates 43 . Cells were cultured in CK+DCI medium with 10 µg/mL Y27 for the first 48 h. Microwell fabrication and culture: Microwell replicate topographies were fabricated by moulding from cell culture surfaces (EZSPHERE TM ) with different microwell width and depth. Briefly, poly(dimethylsiloxane) PDMS (Sylgard TM 184, Ellsworth Adhesives, 10:1 ratio) was mixed with Hexanes (30% vol/vol), and polymerized for 2 h at 80°C. Hydrogel microwell topographies were fabricated through NorHA mixed with 1 mM thiolated RGD and crosslinked with MMP-degradable peptide crosslinkers (320-390 nm, Omnicure S1500, Exfo) for 5 min at 5 mW cm -2 . For primary AT2 culture, mouse lung fibroblasts were encapsulated at 5 million cells/mL (or as otherwise noted). iPSC or primary mouse AT2 cells were added atop with different cell densities and cultured in CK+DCI medium (iAT2) or modified SAGM media as previously described 40 . Briefly, Small Airway Epithelial Cell Growth Basal Media (SABM, Lonza) was mixed with Insulin/Transferrin, Bovine Pituitary Extract, Gentamycin, and Retinoic Acid as well as 0.1 mg/mL Cholera Toxin (Millipore Sigma), 25ng/mL EGF (Peprotech), and 5% FBS. CK+DCI and SAGM media were supplemented with 10 µg/mL Y27 for the first 48 h. In silico modeling: To simulate initial cell seeding within microwells, Cinema 4D (C4D) rigid body dynamic simulations were used. Briefly, microwells were created according to various geometries and tagged as collider bodies, and cells (spheres, 12.5 µm) were tagged as rigid bodies and seeded into wells using simulated gravity. Cells were arrayed above the microwell with random seed points, and after settling, a Boole object was used to segment the simulation in half and rendering was carried out using C4D. Analysis of scRNA-seq data. Reads were aligned and unique molecular identifier (UMI) counts obtained using STAR-Solo (v2.7.9a) 44 . Seurat (v4.0.1) 45 was used for all downstream scRNA-seq analysis. Cells with less than 200 genes, greater than 2 Median absolute deviation above the median, and with potential stress signals of greater than 25% mitochondrial reads were removed. Data was normalized and scaled using the Statistical analysis and reproducibility. GraphPad Prism 9 software was used for statistical analyses. Statistical comparisons between two experimental groups were performed using two-tailed Student's t-tests and comparisons among more groups were performed using one-way or two-way ANOVA with Bonferroni post hoc testing. All experiments were repeated as described in the text. 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