We sincerely thank members of the Wilson, Kotton, and Hawkins laboratories for their helpful discussions. We are also very grateful to Greg Miller (CReM Laboratory Manager) and Marianne James (iPSC Core Manager) for their invaluable support. We thank Brian Tilton and the BUMC Flow Cytometry Core for their technical assistance with cell sorting (supported by NIH grant #1S10OD021587-01A1). This work was supported by a CJ Martin Early Career Fellowship from the Australian National Health and Medical Research Council awarded to RBW; NIH grant F30HL147426 to KMA; an I.M. Rosenzweig Junior Investigator Award from The Pulmonary Fibrosis Foundation and an Integrated Pilot Grant Award through Boston University Clinical &#38; Translational Science Institute (1UL1TR001430) to KDA; Swiss National Science Foundation (P2ELP3_191217) to ABA; NIH grants U01HL148692, U01HL134745, U01HL134766 and R01HL095993 to DNK; and NIH grants U01TR001810, R01DK101510, and R01DK117940 awarded to AAW; iPSC maintenance, banking, and sharing are supported by NIH grant NO1 75N92020C00005. Figure 1 was created using Biorender.com.

All experiments involving the differentiation of human iPSC lines were performed in compliance with the Institutional Review Board of Boston University (protocol H33122). The dermal fibroblasts, procured for reprogramming to iPSCs, were obtained from a donor with written informed consent, under the approval of the Human Research Protection Office of Washington University School of Medicine, St. Louis, MO. Reprogrammed iPSCs were generated at the Center for Regenerative Medicine at Boston University and Boston Medical Center, Boston, MA.
1. Alveolosphere dissociation
<ol>
	<li>Prepare complete serum-free differentiation media (cSFDM) as per the composition mentioned in Table 1.</li>
	<li>Prepare CK + DCI media in the prepared cSFDM base as per Table 2.</li>
	<li>Thaw 2D (human embryonic stem cell-qualified) and/or 3D (growth-factor reduced) matrix on ice as required for the experimental needs.</li>
	<li>Aspirate all the CK + DCI medium using a pipette or aspirating pipette with vacuum from the 3D matrix droplets containing alveolospheres, derived from directed differentiation19, in a 12-well plate.</li>
	<li>Add 1 mL of dispase (2 mg/mL) per droplet. Gently pipette the droplet into the dispase using a P1000 pipette. Incubate at 37 &#176;C for 1 h, pipetting up and down once after 30 min.</li>
	<li>Transfer the dissociated organoids (from Step 1.5) from one matrix droplet in the dispase to a 15 mL conical tube. To wash, add 10 mL of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM, see Table of Materials).</li>
	<li>Centrifuge at 300 x g for 5 min at room temperature. Aspirate the supernatant using a pipette or aspirating pipette with vacuum, leaving as little supernatant as possible. 
	NOTE: It is important to remove all dispase as any remaining dispase may dissolve the matrix that the cells will subsequently be seeded into. If a clear haze is seen above the pellet, the dispase has not completely dissolved the matrix, and more dispase can be added to the pellet for another 20-30 min at 37 &#176;C.</li>
	<li>Resuspend the cells in 1 mL of 0.05% trypsin per droplet and transfer back to the 12-well plate. Incubate at 37 &#176;C for 12-15 min. Observe the dissociation under a microscope. Avoid over-pipetting the cells at this stage. 
	NOTE: At the end of incubation, the cells need to achieve a single-cell suspension after pipetting 3-5 times with a P1000 pipette. For passaging iAT2s to ALI (Step 3), the trypsinization time needs to be minimized (maximum 12 min), such that the cells are in 2-3-cell clumps rather than single-cell suspension when ready for plating onto the cell culture insert.</li>
	<li>Stop the action of trypsin with an equal volume of FBS-containing medium (10% ES-qualified FBS in DMEM). Centrifuge at 300 x g for 5 min at room temperature.</li>
	<li>Wash the cells with 10 mL of IMDM. Centrifuge at 300 x g for 5 min at room temperature.</li>
	<li>Resuspend the cells in an appropriate volume for counting, and then count the cells using a hemocytometer (see Table of Materials). 
	NOTE: From one confluent 50 &#181;L matrix droplet seeded at 400 cells/&#181;L, the expected yield is 500,000 to 1.5 x 106 cells per droplet.</li>
	<li>Use the single-cell suspension of iAT2 cells to generate alveolospheres by plating in the 3D matrix (Step 2) and/or plating on cell culture inserts for ALI culture (Step 3).</li>
</ol>
2. 3D plating of iAT2s
<ol>
	<li>After counting (Step 1.11), determine the number of desired cells to replate in the 3D matrix (400 cells/&#181;L of the matrix with 50-100 &#181;L of 3D matrix droplets per well of a 12-well plate). Centrifuge the cells at 300 x g for 5 min at room temperature. Remove as much supernatant as possible using a pipette.</li>
	<li>Resuspend the cells in the 3D matrix. Resuspend quickly and on ice, if needed, to prevent the matrix from polymerizing (which occurs when warm).</li>
	<li>Use a P200 pipette to dispense one 3D matrix droplet per well into a pre-warmed 12-well plate. Pipette carefully to avoid creating bubbles in the matrix droplet. Do not allow the cell suspension to settle while dispensing multiple droplets.</li>
	<li>Place the plate in a 37 &#176;C incubator for 20-30 min to allow the matrix droplets to polymerize.</li>
	<li>Add 1 mL of CK + DCI + 10 &#181;M of Y-27632 medium (see Table of Materials) per well to cover the matrix droplet.</li>
	<li>After 72 h, change the medium to CK + DCI without 10 &#181;M of Y-27632.</li>
	<li>Replace the medium with fresh CK + DCI every 48-72 h. 
	&#8203;NOTE: iAT2s will typically need to be passaged approximately every 10-14 days, depending on cell line and plating density.</li>
</ol>
3. Passaging of iAT2s to ALI
<ol>
	<li>Prepare freshly coated 6.5 mm cell culture inserts 1 h before use by diluting the 2D matrix in Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) to working solution as per manufacturer&#39;s instructions for the 2D matrix (see Table of Materials). Then, add 100 &#181;L of the diluted matrix per 6.5 mm cell culture insert. Allow the matrix to polymerize in a 37 &#176;C incubator for 30 min or at room temperature for 1 h.</li>
	<li>Aspirate the excess matrix from the cell culture inserts using a pipette or aspirating pipette with vacuum and rinse once with DMEM/F12. Aspirate this wash immediately before adding the cells.</li>
	<li>After counting, determine the number of required cells to seed the desired number of cells in the culture inserts (520,000 live cells/cm2, equivalent to 172,000 live cells per 6.5 mm cell culture insert). Centrifuge the cells at 300 x g for 5 min at room temperature. Remove as much supernatant as possible.</li>
	<li>Resuspend the cells in the appropriate volume to seed with 100 &#181;L of CK + DCI + 10 &#181;M Y-27632 per cell culture insert (cell suspension should be 1,720 cells/&#181;L ). Add 100 &#181;L to the apical compartment of the cell culture insert. Gently agitate the plate in a cross-pattern to ensure an even distribution of cells across the cell culture insert, and confirm this by checking under a microscope at 4x objective. 
	NOTE: Seeding density is critical and may require optimization, ranging from 160,000-300,000 cells per 6.5 mm cell culture insert. Calcein green (see Table of Materials) cell-permeable dye may be added to the cells and viewed on an inverted fluorescent microscope to visualize the cells after seeding.</li>
	<li>Add 500 &#181;L of CK + DCI + 10 &#181;M of Y-27632 to the basolateral compartment of each cell culture insert.</li>
	<li>Aspirate apical CK + DCI + 10 &#181;M of Y-27632 medium 48 h after seeding using a pipette to initiate ALI (known as &#34;air-lift&#34;). 
	NOTE: The cells should be 100% confluent at this point. If the cells are not 100% confluent, Steps 3.7-3.8 should still be performed at the indicated time points; confluent cell monolayers may take longer to form.</li>
	<li>Change the basolateral CK + DCI + 10 &#181;M of Y-27632 medium after 72 h to CK + DCI without 10 &#181;M Y-27632. 
	NOTE: There should be minimal if any, &#34;leak&#34; of medium to the apical side. However, if this does occur in the first few days, continue to aspirate the apical side daily until there is no further leakage.</li>
	<li>Replace the basolateral medium with fresh CK + DCI every 48-72 h. 
	NOTE: The iAT2s used in ALI cultures are maximally matured at 5-14 days post-plating.</li>
	<li>Maintain the cells with careful monitoring for up to 28 days post-plating, if needed, for more prolonged experimentation. 
	NOTE: Visible signs of ALI failure, such as peeling of the cells away from the edge of the insert or formation of holes in the monolayer, may be observed after an overextended time in culture, at which point the ALI is no longer usable for experiments.</li>
</ol>

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The authors have nothing to disclose.

AT2s maintain lung homeostasis, and dysfunction of these key alveolar cells can both cause and result from various lung diseases. Due to the difficulty of accessing and isolating primary human AT2s, iAT2s are generated. By applying the directed differentiation methods described elsewhere25 and the expansion and cell seeding described here, iPSCs generated from any individual can be differentiated into robustly self-renewing iAT2s, thus providing patient-specific cells for biomedical research, including basic biomedical research developmental studies, disease modeling, cell-based therapies, or drug screens. The present protocol details a method for dissociating iAT2s to a single-cell suspension, which can then be replated in 3D Matrigel to generate alveolospheres for cell expansion or replated in 2D ALI culture for further experiments.
The protocol has several critical steps to ensure successful passaging and replating in both 3D and ALI cultures. Mechanical trituration must be minimized, as excessive pipetting can decrease cell viability. In addition, the seeding density of iAT2s in both 3D and 2D ALI cultures is critical; for 3D culture, in general, 400 cells/&#181;L of 3D Matrigel is optimal for most iPSC lines. However, a range of densities from 100-500 cells/&#181;L has, at times, been successful, and optimizing the density may be required for different cell lines. For ALI culture, optimizing the seeding density of iAT2s on the cell culture inserts is also essential to achieve a confluent monolayer of cells 48 h after seeding (i.e., on the day of air-lift). Some iAT2 lines require more cells per insert; thus, if troubleshooting is required, a range from 160,000-300,000 cells/insert for 24-well cell culture inserts is recommended. 3D cultures can also be scaled to 24- and 48-well plates by maintaining the seeding density at 400 cells/&#181;L of 3D matrix and reducing matrix droplet size to 25 &#181;L and 20 &#181;L, respectively. ALI cultures can be scaled to 96-well cell culture inserts by coating each insert with 30 &#181;L of the matrix, plating 80,000 cells in 30 &#181;L per insert, and feeding with 150 &#181;L of basolateral media per well. Seeding density and matrix and media volumes need to be scaled and optimized accordingly for other plate formats.
A limitation of this protocol is that the cells used for ALI plating have to be sufficiently purified to be NKX2-1+ and SFTPC+19,20,21,23 to form iAT2 ALIs, and ALI culture formation may vary from line to line. In addition, this protocol allows expansion and generation of AT2-only cultures, providing a reductionist model system based on a single key alveolar cell type. Importantly, other relevant alveolar cell types, such as alveolar type 1 cells, were missing from these platforms. Other groups have had success culturing primary human AT2s as spheroids or organotypic cultures with or without fibroblasts1,14,15,16,17; however, primary human AT2s tend to lose their expression of key surfactants when cultured in normal 2D culture without ALI conditions26. Furthermore, recent work has shown that iAT2s express higher proliferative markers and lower AT2 maturation markers than fresh, uncultured human primary AT2s27. Despite these differences between iAT2s and fresh primary human AT2s, we found that even cultured primary AT2s displayed transcriptomic differences from fresh, uncultured primary AT2s27, and thus conclude that these in vitro models have various strengths and limitations for modeling in vivo lung biology.
The iAT2 platform can be applied to study genetic mutations from patient-derived iPSCs19,20. The system could also be substantially scaled up to accommodate high throughput drug screening. In addition, iAT2 ALIs are suitable for environmental exposures such as viral or bacterial infection or exposure to cigarette smoke, e-cigarette vapor, or other aerosols23. In summary, this protocol for generating both 3D and 2D ALI cultures of iAT2s allows for long-term culture of a disease-relevant cell type and provides a physiological, scalable platform that enables the usage of these cells in many applications.

In the lung, the airway and alveolar epithelial cells are the first to encounter inhaled environmental exposures, including pathogens transmitted via inhaled aerosols and noxious stimuli such as cigarette smoke. Type 2 alveolar epithelial cells (AT2s) are essential in maintaining lung homeostasis as they are the facultative progenitors of the distal lung epithelium, produce surfactants to relieve alveolar surface tension, and mount the innate immune response to inhaled exposures1,2. However, AT2 dysfunction can result from lung injuries or mutations in genes that are selectively expressed in AT2s, such as SFTPC, SFTPB, and ABCA33,4,5. Previous approaches for studying these genetic mutations have relied on mouse models6 or engineered, mutation-containing vectors introduced into immortalized cell lines7. Therefore, platforms that can model the effects of genetic and environmental perturbations on AT2s in the physiologically relevant cell types and in a productive in vitro system are needed to understand further the role AT2s play in health and disease.
In terms of modeling airborne exposures, air-liquid interface (ALI) cultures of primary airway epithelial cells have been successfully utilized to reveal key molecular responses to cigarette smoke8 and to model airway infection9,10. A comparable ALI culture system for the alveolar epithelium, a key site of infection or injury in disease, is much less developed compared to the airway epithelial model system. Immortalized cell lines have been cultured at ALI as a proxy for the alveolar epithelium11,12, but these cell lines are transcriptomically distinct from primary alveolar epithelial cells13 and lack key cellular machinery, such as the ability to secrete surfactants or form tight junctions at ALI12. Primary human AT2s can be cultured in 3D spheres in the presence of fibroblasts1,14 but are subject to limitations, including the limited accessibility from explant lungs and their tendency to senesce or lose cellular phenotype in most cultures to date. These characteristics present barriers to the widespread adoption of primary AT2 in vitro studies, although recent progress has been made in optimizing feeder-free 3D cultures for the expansion of primary AT2s15,16,17,18.
Directed differentiation protocols have been developed to recapitulate in vivo developmental milestones to generate human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s)19. iAT2s grow as self-renewing spheres in 3D serum-free culture in a defined medium containing CHIR99021, KGF, dexamethasone, cAMP, and 3-isobutyl-1-methylxanthine (IBMX), termed &#34;CK + DCI&#34;19, in the absence of fibroblast feeders and can be cultured for &#62;20 passages20,21. Moreover, iAT2s share a transcriptomic program with human adult primary AT2s, form lamellar bodies, and produce and package surfactant19,21,22. This protocol details the serial passaging of iAT2s by dissociating the cells to a single-cell suspension. At this point, iAT2s can be replated and expanded further in 3D culture or plated in 2D ALI culture23. These methods can be used to study the intrinsic biology of AT2s in homeostasis and disease20,22 and to interrogate the effects of compounds or stimuli in a scalable, physiologically relevant platform23,24, as has previously been shown.

<table><tbody><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma</td><td>I5879</td><td>For CKDCI</td></tr><tr><td>12 Well Cell Culture Plate</td><td>Corning</td><td>3513</td><td>Plates for culturing</td></tr><tr><td>1-Thioglycerol (MTG)</td><td>M6145</td><td>Sigma</td><td>For CKDCI</td></tr><tr><td>2D Matrigel (matrix) - Matrigel hESC-Qualified Matrix</td><td>Corning</td><td>8774552</td><td>To coat ALI Transwells</td></tr><tr><td>3D Matrigel (matrix) - Growth Factor Reduced Matrigel</td><td>Corning</td><td>356230</td><td>To grow iAT2 spheres in</td></tr><tr><td>8-bromoadenosine 30,50-cyclic monophosphate sodium salt (cAMP)</td><td>Sigma</td><td>B7880</td><td>For CKDCI</td></tr><tr><td>Ascorbic Acid</td><td>A4544</td><td>Sigma</td><td>For CKDCI</td></tr><tr><td>B27 w/out retinoic acid</td><td>Life Technologies</td><td>12587-010</td><td>For CKDCI</td></tr><tr><td>Bovine Serum Albumin (BSA) (7.5%)</td><td>Thermo Fisher</td><td>15260037</td><td>For CKDCI</td></tr><tr><td>Calcein blue</td><td>Life Technologies</td><td>C1429</td><td>For live/dead discrimination in flow cytometry</td></tr><tr><td>Calcein green</td><td>Life Technologies</td><td>C1430</td><td>Optional visualisation of cells on cell culture insert</td></tr><tr><td>Cell culture inserts - Costar 6.5 mm Clear Transwells with 0.4 &amp;micro;m pore size</td><td>Millipore-Sigma</td><td>CLS3470-48EA</td><td>ALI transwells</td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423</td><td>For CKDCI</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>For CKDCI</td></tr><tr><td>Dispase (2 mg/mL)</td><td>Thermo Fisher</td><td>354235</td><td>To dissolve matrigel</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>11995</td><td>To make trypsin inactivation media (10% FBS in DMEM)</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)</td><td>Thermo Fisher</td><td>A4192002</td><td>To wash</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (PBS)</td><td>Thermo Fisher</td><td>14190</td><td>For flow cytometric analyses</td></tr><tr><td>Fetal bovine serum (FBS) (Hyclone charaterized)</td><td>GE Healthcare Life Sciences</td><td>SH30071.03</td><td>To make trypsin inactivation media (10% FBS in DMEM)</td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050-061</td><td>For CKDCI</td></tr><tr><td>Ham&#039;s F-12 Nutrient Mixture (F12)</td><td>Cellgro</td><td>10-080-CV</td><td>For CKDCI</td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>02-671-6</td><td>For cell counting</td></tr><tr><td>Invivogen&amp;nbsp;Primocin 1 G (50 mg/mL)</td><td>Fisher Scientific</td><td>NC9392943</td><td>For CKDCI</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Thermo Fisher</td><td>12440053</td><td>For CKDCI</td></tr><tr><td>Millicell ERS-2 Voltohmeter</td><td>Millipore</td><td>MERS00002</td><td>To measure trans-epithlial electrical resistance</td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; supplement</td><td>Life Technologies</td><td>17502-048</td><td>For CKDCI</td></tr><tr><td>Recombinant human KGF</td><td>R&amp;amp;D Systems</td><td>251-KG-010</td><td>For CKDCI</td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td>For CKDCI</td></tr><tr><td>Trypan blue</td><td>Thermo Fisher</td><td>15250061</td><td>For cell count during passaging</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Gibco</td><td>25-300-062</td><td>To dissociate iAT2 spheres</td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris</td><td>1254</td><td>Add to cells after passaging</td></tr></tbody></table>

generating 3d spheres and 2d air-liquid interface cultures of human induced pluripotent stem cell-derived type 2 alveolar epithelial cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The iAT2s were passaged to a single-cell suspension and then replated in 3D matrix or in 2D on cell culture inserts for ALI culture (Figure 1). After single-cell dissociation, the iAT2s were analyzed by flow cytometry. Briefly, the cells resuspended in 1% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) with calcein blue viability dye (1:1000) were analyzed on a flow cytometer, gating for non-fragments, singlets, and tdTomato. Here, as an example, is the SPC2 line (SPC2-ST-B2 clone20), which has a tdTomato reporter targeted to the endogenous SFTPC locus for ease of visualization and tracking of the AT2 program over time in culture (Figure 2A). The iAT2 SFTPC-tdTomato expression was maintained when replated in the 3D matrix as spheres (Figure 2B) and when plated on cell culture inserts (Figure 2C). Trans-epithelial electrical resistance (TEER) can be measured to determine the integrity of the ALI culture (Figure 2D). To measure TEER, a voltohmmeter was used (see Table of Materials), with 100 &#181;L of CK + DCI medium added to the apical chamber. The cell culture inserts coated with Matrigel in the absence of seeded cells were treated as blanks. Readings were taken at three locations in every well.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63875/63875fig01v2.jpg" /> 
Figure 1: Schematic representation of the protocol workflow. <a href="https://www.jove.com/files/ftp_upload/63875/63875fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63875/63875fig02.jpg" /> 
Figure 2: Representative results of the protocol. (A) Representative flow cytometry results for SFTPC-tdTomato in human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s) cultured in 3D. The negative control shown is non-lung endoderm (CD47lo). (B) Representative live-cell imaging of iAT2s cultured in 3D at various days post passage (dpp) (SFTPC-tdTomato, scale bar = 50 &#181;m). (C) Representative live-cell imaging of iAT2s plated at air-liquid interface (SFTPC-tdTomato, scale bar = 500 nm). (D) Representative trans-epithelial electrical resistance of iAT2s plated at the air-liquid interface. n = 3, error bars indicate standard deviation. <a href="https://www.jove.com/files/ftp_upload/63875/63875fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume for 500 mL</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>375 mL</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture (F12)</td>
			<td>125 mL</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>B-27 (with RA) supplement</td>
			<td>5 mL</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>2.5 mL</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>BSA (7.5% stock)</td>
			<td>3.3 mL</td>
			<td>0.05%</td>
		</tr>
		<tr>
			<td>Primocin (50 mg/mL stock)</td>
			<td>1 mL</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Glutamax (100x stock)</td>
			<td>5 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Ascorbic Acid (50 mg/mL stock)</td>
			<td>500 &#181;L</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>1-Thioglycerol (MTG) (from 26 &#181;L in 2 mL IMDM)</td>
			<td>1.5 mL</td>
			<td>4.5 x 10-4 M</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the complete serum-free differentiation media (cSFDM).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>3 &#181;M</td>
		</tr>
		<tr>
			<td>rhKGF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>50 nM</td>
		</tr>
		<tr>
			<td>8-bromoadenosine 30,50-cyclic monophosphate sodium salt (cAMP)</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>0.1 mM</td>
		</tr>
	</tbody>
</table>
Table 2: Compositions of the CK + DCI media.

Watch this Scientific Journal Video about Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells at JoVE.com

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

generating-3d-spheres-2d-air-liquid-interface-cultures-human-induced

Research

JoVE Journal

Developmental Biology

4.7K Views.  Boston University and Boston Medical Center. Dit protocol schetst een methode voor het kweken en doorgeven van een belangrijke celpopulatie in de longen en vormt een fysiologisch relevant platform voor blootstelling aan het milieu. Het grote voordeel van deze techniek is dat het kan worden gebruikt om longbiologie te onderzoeken in de context van homeostase en ziekte. Deze methode kan inzichten geven, met name in longziekten die longblaasjes aantasten, waaronder die veroorzaakt door genetische varianten, blootstelling aan het milieu, in...

Video: Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Bioengineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Medicine

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Biology

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.

1. Preparation and Maintenance of 3D Sphere Cocultures

	Dissociate hAstros from 3D aggregates in suspension.&#8203;
	
		Gently dissociate human astrocyte ...

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

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