*Article* **A New Perspective for Bone Tissue Engineering: Human Mesenchymal Stromal Cells Well-Survive Cryopreservation on** β**-TCP Scaffold and Show Increased Ability for Osteogenic Differentiation**

**Liudmila Leppik 1,\*, Anna Gempp 1, Zyrafete Kuçi 2, Selim Kuçi 2, Peter Bader 2, Halvard Bönig 3, Ingo Marzi <sup>1</sup> and Dirk Henrich <sup>1</sup>**


**Abstract:** The clinical breakthrough of bone tissue engineering (BTE) depends on the ability to provide patients routinely with BTE products of consistent pharmacological quality. The bottleneck of this approach is the availability of stem cells. To avoid this, we suggest immobilization of randomdonor-derived heterologous osteoinductive MSCs onto osteoconductive matrices. Such BTE products could then be frozen and, after thawing, could be released as ready-to-use products for permanent implantation during surgery. For this purpose, we developed a simple protocol for cryopreservation of BTE constructs and evaluated the effects of this procedure on human MSC (hMSCs) metabolic and osteogenic activity in vitro. Our findings show that hMSCs can be freeze-thawed on a β-TCP scaffold through a technically simple procedure. Treated cells sustained their metabolic activity and showed favorable osteogenic potential. Mechanistically, HIF1α and YBX1 genes were activated after freeze-thawing, and supposed to be linked to enhanced osteogenesis. However, the detailed mechanisms as to how the cryopreservation procedure beneficially affects the osteogenic potential of hMSCs remains to be evaluated. Additionally, we demonstrated that our BTE products could be stored for 3 days on dry ice; this could facilitate the supply chain management of cryopreserved BTE constructs from the site of manufacture to the operating room.

**Keywords:** human MSCs; cryopreservation; osteogenic differentiation; scaffold; 3D culture

#### **1. Introduction**

Large non-healing bone defects, largely irrespective their etiology, constitute a major challenge for patients and physicians [1–3]. Treatment of choice is with autologous bone grafts [4]; however, several other treatments are also used [5–7]. These methods are not always effective, but even where they are, the morbidity associated with autologous tissue harvesting is considerable, fueling the search for alternatives.

Bone tissue engineering (BTE) is one such alternative approach, which holds great promise for promoting bone healing and regeneration while overcoming some of the drawbacks of current techniques. BTE approaches typically combine bone forming stem or progenitor cells with scaffolds, which restore missing bone volume and signaling molecules, which control cell–cell and cell–scaffold interactions in the bone defect. Among many different materials used to construct scaffolds, the biodegradable ceramic scaffolds with desirable

**Citation:** Leppik, L.; Gempp, A.; Kuçi, Z.; Kuçi, S.; Bader, P.; Bönig, H.; Marzi, I.; Henrich, D. A New Perspective for Bone Tissue Engineering: Human Mesenchymal Stromal Cells Well-Survive Cryopreservation on β-TCP Scaffold and Show Increased Ability for Osteogenic Differentiation. *Int. J. Mol. Sci.* **2022**, *23*, 1425. https://doi.org/ 10.3390/ijms23031425

Academic Editor: Carlo Ventura

Received: 16 December 2021 Accepted: 25 January 2022 Published: 26 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pore size, porosity, and mechanical properties are probably the most preferable in BTE, since these materials generally show better tissue responses compared to polymers and metals [8]. Tricalcium phosphate (TCP) ceramics in contrast to other ceramics (for example hydroxyapatite) found wide application in the clinic due to their osteoconductivity and bioresorbability [9]. Clinical BTE approaches have demonstrated encouraging early outcomes [10]. However, in these types of approaches availability of stem cells is a bottleneck of the whole procedure. Isolation, characterization, and expanding of autologous mesenchymal stem cells (MSCs) or use of cryopreserved dissociated stem cell populations from cryobanks [11] need significant investment of time and resources that limits translation of BTE approaches into clinic. To be able to provide BTE products of consistent medicinal quality, conducive to routine clinical use, we proposed to immobilize random-donor-derived osteoinductive MSCs onto osteoconductive matrices. These BTE products were then frozen and released ready-to-use for permanent implantation during surgery after thawing.

The work builds on earlier work which has definitively established that cryopreserved MSCs maintain potential for proliferation and osteogenic differentiation in vitro [12–15] and in vivo [16,17]. The methods used, however, were not conducive to clinical application. A recent pilot study of Hernandez–Tapia showed that osteoblasts have good survival rate after cryopreservation on β-TCP scaffold [18]. The equally recent study of Mutsenko et al. [19] showed that MSCs could be frozen in 3D collagen-hydroxyapatite scaffold without significant loss of cell viability. Although these studies use technically challenging methodology, they represent invaluable first steps towards the creation of a tissue-engineered constructs biobanks.

In the present study, we developed a simple protocol for cryopreservation of BTE constructs (human MSCs seeded on 3D β-TCP scaffold) and evaluated the effects this procedure has on human MSCs metabolic and osteogenic activity in vitro.

#### **2. Results**

#### *2.1. Effect of Cryopreservation on hMSCs Metabolic Activity*

First, we analyzed how cryopreservation affects the metabolic activity of cells frozen on scaffold granules following common freezing protocol and whether the cells could recover their activity with time. Our results show that MSCs retained approximately 40% of their metabolic activity at first two days after cell thawing as compared to control (not frozen) cells (Figure 1A), and at later time points cells increased their activity to the level of control cells. We also compared recovery of cells frozen in this way with the cells frozen by mean of "air-dry" freezing protocol. No significant difference in metabolic activity of these two groups of cells post-thaw was detected for all time points (Figure 1B). Based on this the "air-dry" protocol was chosen for the following experiments.

In addition, we analyzed if "air-dry" 3D cryopreserved hMSCs could be stored at −20 ◦C, in dry ice or in a combination of both. We compared the metabolic activity of cryopreserved cells stored in (1) liquid nitrogen, (2) liquid nitrogen and −20 ◦C, (3) liquid nitrogen and dry ice (3 days), and (4) liquid nitrogen, dry ice (3 days) and −20 ◦C (1 day) after thawing. Our results show that cells did not survive storage at −20 ◦C (Figure 1D); however, storage in dry ice for 3 days did not negatively affect cell metabolic activity after thawing. The combination of both storage conditions, dry ice, and −20 ◦C had a significant negative effect on cell metabolic activity at 2 and 7 days after thawing (Figure 1D).

To compare the cell distribution on a scaffold before and after freezing, live cells were visualized with Calcein AM and DAPI staining. As can be seen from Figure 2, there were less cells present on a granule surface and more cells present inside pores in the samples 1-h post-thaw (Figure 2C,D) as compared to control samples without freezing.

#### *2.2. Cryopreserved hMSCs Show Stronger Osteogenic Activity Than Control Cells*

In order to analyze if cryopreservation has effect on osteogenic potential of hMCS post-thawing and therefore on future use of these cells in BTE approaches, cells after cryopreservation were thawed and cultured for two weeks in osteogenic-supplemented medium

(Figure 1C). There was no difference in the level of alkaline phosphatase (ALP) expression among control and cryopreserved cells at day 7 of culture in osteogenic-supplemented medium. In both type of cells expression of ALP was significantly higher than in cells cultured in growth medium (Figure 1E).

The results of osteogenic gene expression analysis are presented in Figure 3. Surprisingly, the expression of all tested genes was enhanced in the experimental group as compared to control (not frozen) cells. Expression of early markers of osteogenesis RunX2, Col1, and ALPL in the experimental group peaked on day 7 of osteogenic culture, whereas in the control group it did not peak until day 14. Col1 expression was significantly higher (*p* < 0.05) in experimental group cells as compared to control cells at day 7 of osteogenic culture. On the contrary, its expression was significantly (*p* < 0.05) higher in control group cells at day 14 of culture. Expression of the SPP1 gene was low in both groups of cells on day 7, but was significantly increased on day 14 in the experimental group as compared to control group cells (*p* < 0.01). None of osteogenic marker genes was expressed in cells cultured in growth medium except for SPP1, which showed strong up regulation on day 14 of culture (Supplementary Figure S1). In the control-DMSO group, in which cells were treated with freezing medium but were not frozen, expression of osteogenic marker genes was similar as in control cells except for Col1. This gene was highly upregulated in these cells at day 7 of osteogenic culture (Supplementary Figure S1).

**Figure 1.** hMSCs preseeded on scaffolds retain their metabolic activity after cryopreservation. (**A**) Metabolic activity of MSCs was measured 1, 2, 4, and 7 days after cryopreservation, shown in % of control cells metabolic activity. MSCs were preseeded on scaffolds 48 h prior to cryopreservation. (**B**) There was no significant difference in metabolic activity of hMSCs processed with two different cryopreservation protocols and measured 2 days after cryopreservation. (**C**) Metabolic activity of control and experimental cells at 7 and 14 days of culture in osteogenic supplemented medium showed no significant differences. (**D**) hMSCs were first cryopreserved in liquid nitrogen and either stored for 3 days on dry ice or stored for 3 days on dry ice and 1 day at −20 ◦C. The metabolic activity of cells was measured at days 2 and 7 post-thaw and is shown in % of metabolic activity of cells stored in liquid nitrogen only. (**E**) ALP expression is significantly enhanced in both, control and experimental group cells at day 7 of culture in osteogenic-supplemented medium, as compared to cells cultured in growth medium. (\* *p* < 0.05, § *p* < 0.01).

**Figure 2.** hMSCs distribution on a β-TCP scaffold granule before and post-thawing. Control hMSCs were seeded on a β-TCP scaffold and cultured either for 24 (**A**) or 72 h (**B**). Experimental hMSCs were seeded on a β-TCP scaffold, cultured for 24 h, frozen in liquid nitrogen for seven days, thawed, and cultured for 1 (**C**) and 48 (**D**) hours. All cells are stained with Calcein AM and DAPI. Dotted line shows the edges and pores of β-TCP granule. (5× magnification; scale bar = 100 μM).

**Figure 3.** Osteogenic marker genes mRNAs expression in control and experimental cells. Expression of osteogenic marker genes was evaluated in experimental and control group hMSCs at days 7 and 14 of culture in osteogenic-supplemented medium. (\* *p* < 0.05; \*\* *p* < 0.01).

#### *2.3. Cryopreservation Has an Effect on Expression of Hypoxia-Related Genes*

Hypothesizing that the positive effect of cryopreservation on hMSCs osteogenic differentiation could be promoted by the difference of oxygen concentration developed due to temperature difference, we compared expression of hypoxia-related genes in experimental and control cells (Figure 4). The results showed that expression of the major hypoxia marker gene HIF1α and several of its target genes was affected by cryopreservation (Figure 4 and Supplementary Figure S2). Expression of HIF1α in the experimental group cells was significantly (*p* < 0.05) higher than in the control cells one day post-thaw. On day seven, expression of HIF1α and its target genes PDK1, SLC2A1, EGLN1, and BNIP3 was higher in cells which had undergone the cryopreservation procedure compared to control cells, albeit not significantly. Expression of VEGF mRNA was significantly lower on days 1 and 7, and significantly higher on day 14 of culture in the experimental group cells as compared to controls (Figure 4A).

**Figure 4.** mRNA levels of gene expression of HIF1-α, YBX1, and VEGF and IL-6 protein secretion at days 1, 7, and 14 of culture. (**A**) Expression of HIF1α, YBX1, and VEGF genes was evaluated in experimental and control group hMSCs at days 1, 7, and 14 of culture in osteogenic-supplemented medium. (**B**) Concentration of IL-6 was measured in medium supernatants of both groups at days 1, 2, 3, and 7 of culture. (\* *p* < 0.05).

#### *2.4. Cryopreservation Enhances YBX1 Expression but Has No Effect on Wnt/Smad Pathways*

We analyzed if the freezing procedure has an effect on expression of the cold shock protein YBX1, known to react to low temperature and to be an important regulator of transcription [20]. Expression of this gene was significantly (*p* < 0.05) enhanced as early as 1 day after thawing in the experimental group and remained high until day 7 of culture. On day 14 of culture, expression of this gene was the same in both experimental and control group cells (Figure 4).

In order to investigate if the freezing procedure could have effect on key signaling (Wnt and SMAD) pathways in hMSCs and thereby influence hMSCs osteogenic potential, we analyzed expression of Wnt3a, SMAD5, MAPK8, and MAPK14 genes early after the freezing and thawing event (1 day) as well as at later time points (7 and 14 days). No significant difference in expression of these genes among experimental and control group cells was found at any time point (Supplementary Figure S3).

#### *2.5. Effect of Cryopreservation on Interleukin Expression*

We evaluated if cryopreservation has an effect on hMSC's IL-10 and IL-6 expression 1–7 days into osteogenic culture, as the mediators could be important for future use of these cells in BTE approaches (Figure 4B). In both control and experimental cells, expression of IL-10 was below detection level of our method (data not shown). Concentration of IL-6 was lower in the experimental group on days 1, 2, and 3 as compared to control-group medium supernatant. On day 7 of osteogenic culture, the difference in expression of IL-6 between two groups of cells lost statistical significance.

#### **3. Discussion**

The clinical breakthrough of bone tissue engineering depends on the ability routinely to provide patients with BTE products of consistent pharmacological quality. For this reason, the ability to batch-produce and cryogenically preserve tissue engineered construct represents major progress. Cryopreservation is the use of very low temperature to maintain living cells and tissues in a quiescent status for a long period, without losing their structure and function [21]. Several works have been devoted to the cryopreservation of engineered biological constructs [18,19,22–24], which could guide our experimentation on the cryopreservation of biofabricated BTE constructs.

In the present study we evaluated whether hMSCs retain their activity after cryopreservation on β-TCP scaffolds. Our results showed that despite the blunted metabolic activity at early time points after thaw and presumably representing a modicum of cell attrition, hMSCs quantitatively regain their metabolic activity later. This temporal loss of metabolic activity could be explained by the loss of cells from the scaffold granule surface, as was shown by Calcein and DAPI staining of cells one-hour post-thaw (Figure 2C, Supplementary Figure S4). These findings are in accordance with other studies, showing that *Callithrix jacchus* MSCs and cells of human osteoblastic cell lines sustain their activity after being frozen on hydroxyapatite or β-TCP scaffolds, respectively [18,19]. In contrast to both these studies, we employed the simplest cell culture and cryopreservation procedures in order to develop protocols which can be later easily translated to clinical application. Therefore, we used a commonly used β-TCP scaffold, MSCs generated from pooled bone marrow mononuclear cells of eight third-party donors and an "air-dry" cryopreservation protocol, which involved the use of standard cell culture equipment and reagents. We also evaluated whether 3D-cryopreserved hMSCs could survive storage at −20 ◦C, dry ice, or a combination of both. Our results clearly demonstrate that cells seeded on β-TCP scaffold and stepwise cryopreserved could not be stored at −20 ◦C as that dramatically effects metabolic activity of cells post-thaw. However, cryopreserved BTE constructs could be stored at least for 3 days on dry ice without an apparent impact on post-thaw cell activity. Cryopreserved hMSCs-coated matrix also survived the storage on dry ice followed by 1 day at −20 ◦C, albeit with significant loss of metabolic activity post-thaw. This information will guide the supply chain management of cryopreserved BTE constructs from the site of manufacture to the operating room.

Osteogenic potential is arguably one of the most important characteristics of biofabricated BTE constructs; therefore, we evaluated how cryopreservation on 3D scaffold affects hMSCs osteogenic potential. Our results showed that osteogenic potential of frozen cells was rather improved. ALP activity of control and experimental cells was the same at day 7 of osteogenic culture (Figure 3). According to the osteogenic marker gene expression analysis, hMSCs osteogenic differentiation was accelerated after freezing/thawing procedure, as most of the analyzed markers were expressed earlier in experimental cells than in control cells. It has been previously shown that MSC retain osteogenic differentiation ability after cryopreservation [25,26], but for hMCSs cultured and cryopreserved on β-TCP scaffold this was not shown before. The effect of increased osteogenic potential was not observed in our "DMSO-control" cells, demonstrating that increased osteogenic gene expression is rather induced by physical effects and not by components of the cryopreservation medium (e.g., DMSO).

We hypothesized that increased osteogenic gene expression could either be a result of unspecific up-regulation of major pathways due to the cryopreservation procedure and/or reaction to the oxygen gradient induced by temperature changes [27,28]. To validate this hypothesis, we analyzed expression of key signaling stem cell fate marker genes and expression of hypoxia-related genes. Results of this analysis showed that expression of Wnt3A, MAPK8, MAPK14, and SMAD5 was not significantly altered in cryopreserved cells (Supplementary Figure S1), whereas the expression pattern of hypoxia related genes differed between control and experimental cells. Expression of HIF1α gene and its target genes PDK1, SLC2A1, EGLN1, and BNIP3 was upregulated in experimental group cells on day 7, whereas in control cells those genes were only upregulated on day 14 of culture. It was previously shown that hypoxia promotes osteogenesis of human MSCs in a HIF-1 dependent manner [29]. We speculate that the cryopreservation procedure affects HIF1α expression, and this mechanism stimulates osteogenesis. In addition, VEGF, a known target of HIF1α, was also significantly upregulated in experimental group cells (Figure 4). A previous study had shown that RunX2 acts together with HIF1α to stimulate angiogenic gene expression in bone cells [30]. Our results are in accordance with it.

We also verified if transcription of cold shock protein YBX1, known to regulate expression of HIF1α [31], was activated after cryopreservation (Figure 4). YBX1 protein possess a cold shock domain and stimulates translation of proteins under low temperature [20,32]. YBX1 protein was shown to be a critical regulator of HIF1α expression in sarcoma cells [33]; however, less is known about the role of this protein in MSCs. Our results show that cryopreservation affects YBX1 gene expression in hMSCs, which would be compatible with a role in the response of the MSCs to cryopreservation and enhancement of their osteogenic potential.

#### **4. Materials and Methods**

#### *4.1. hMSCs Culture and Seeding on* β*-TCP Granules*

Human bone marrow MSC cells (hMSCs) were provided by Department for Children and Adolescents, Division for Stem Cell Transplantation and Immunology, University Hospital Frankfurt. The MSCs were generated from pooled, previously isolated, and cryopreserved mononuclear cells from eight random bone marrow donors by plastic adherence, which have been expanded to near-confluence and cryopreserved in small aliquots as described in detail elsewhere [34]. It has already been demonstrated that these MSCs were effective in the treatment of patients with acute steroid-resistant acute GvHD [35,36]. For subcultures, 3.5 × 105 hMSCs were seeded in T175 cell culture flasks (Sarstedt, Nümbrecht, Germany) and cultured in animal-free growth medium (10% CruxRufa Media Supplement; (TrinovaBiochem, Giessen, Germany); 1 IU/mL of heparin (Ratiopharm, Ulm, Germany); GlutaMax Gibco™ DMEM, (Thermofischer, Dreieich, Germany)) at 37 ◦C, 5% CO2 in a humidified incubator until 80–90% confluence. The culture medium was changed every three to four days and the cells were expanded until passage 5. These MSCs were used either for experiments or stored in liquid nitrogen for future use. Before cryopreservation, hMSC was assessed for expression of typical MSC cell surface markers by using flow cytometry analysis. Phenotypical analysis showed that these cells express high levels of surface proteins CD90 and CD105, but very low or no expression of hematopoietic cell markers CD45 and CD34 (data not shown [34,37]).

For 3D hMSC culture, 500 μL of β-TCP scaffold granules (ChronOS Granules, 1.4–2.8 mm, 60% porosity; Synthes, Oberdorf, Switzerland) were placed in 6.5-mm Transwell ® membrane inserts (3.0 μm pore, polycarbonate, Corning, Wiesbaden, Germany) allocated in individual wells of a 24-well plate. Then 2 × <sup>10</sup><sup>5</sup> hMSCs in 150 <sup>μ</sup>L of PBS (Gibco) were dripped slowly on the scaffolds and incubated for 10 min at 37 ◦C. After incubation, the cell suspension not absorbed by the scaffold was removed, dripped again over the material, and incubated for another 10 min. This step was repeated twice. Afterwards, 1 mL of pre-warmed growth medium was added to each well and cells were further incubated at 37 ◦C, 5% CO2 in a humidified incubator for 24 h.

#### *4.2. Effect of Cryopreservation on Cell Activity*

In order to evaluate the effect of cryopreservation on cell metabolic activity, osteogenic differentiation, and interleukin expression, the scaffold granules with seeded cells were frozen, thawed, and cultured for two weeks in either growth or osteogenic-supplemented medium (experimental group). In the control group, cells were treated the same way as in experimental group except for freeze-thawing.

#### *4.3. Cryopreservation and Thawing of hMSCs on β-TCP-Scaffold*

For "standard protocol" cryopreservation, scaffold granules seeded with cells and cultured for 24 h were transferred into a cryovial, and entire culture medium was exchanged with 1 mL of cold fresh-prepared freezing medium (10% *v*/*v* DMSO, 25% human AB serum (Sigma–Aldrich, Heidelberg, Germany) in DMEM Glutamax). The cryovial with cells was stored at −<sup>80</sup> ◦C for 24 h in a controlled rate freezing container (Nalgene ® Mr. Frosty, Merck, Darmstadt, Germany) and then transferred into a liquid nitrogen for at least 7 days' storage. For thawing, a cryovial with hMSCs-seeded β-TCP scaffold granules was removed from the nitrogen tank, briefly warmed in a water bath (37 ◦C) for 1 min. The cryovial content was transferred into one well of a 24-well-plate and the entire freezing medium was aspirated. Cells were washed with 1 mL of fresh, pre-warmed growth medium, and cultured in 1 mL of growth medium (at 37 ◦C; 5% CO2) for 48 h at which time the cell metabolic activity was assayed.

For the "air-dry protocol" cryopreservation [19], scaffold granules seeded with cells were transferred into a cryovial and the entire culture medium was exchanged with 1 mL of cold fresh-prepared freezing medium. After 15 min incubation on ice, medium was soaked out and cryovial with cell-coated scaffold (without medium) was first frozen at 80 ◦C in a controlled rate freezing container for 24 h, and subsequently stored in liquid nitrogen for at least 7 days. For thawing, a cryovial with hMSCs-seeded β-TCP scaffold granules was removed from the nitrogen tank, briefly warmed in a water bath (37 ◦C) for 1 min. One mL of pre-warmed growth medium was quickly added and the complete cryovial content was transferred into one well of a 24-well-plate. Cells were incubated at 37 ◦C, 5% CO2 for 48 h before cell metabolic activity was assayed.

In order to control for possible effects of DMSO in freezing medium on cell activity after thaw, cells in "DMSO control" group were treated the same way as described in "air-dry protocol", but the freezing step (−80 ◦C, 24 h, and liquid nitrogen storage) was omitted.

#### *4.4. Effect of Storage Temperature*

To analyze if cryostorage temperature has an effect on the functionalized β-TCP scaffold, cryovials with 3D cryopreserved cells were first stored in liquid nitrogen (7 days) and then either directly thawed and cultured or stored for 1 day at: −20 ◦C (freezer); for 3 days in dry ice (Styrofoam container filled with dry ice = −78.5 ◦C); or for 3 days in dry ice and 1 day at 20 ◦C. Thereafter, cells were thawed as described and cultured for 48 h or 7 days before metabolic activity of cells was measured.

#### *4.5. Cell Metabolic Activity Measurements*

Metabolic activity of cells cultured on scaffold granules was measured by means of alamarBlue assay (Bio-Rad, Germany) according to the manufacturer's protocol 48 h after thaw in experimental or 72 h after seeding in control groups, respectively. Briefly, growth medium was soaked out and 400 μL of fresh growth medium and 40 μL of alamarBlue reagent were added to the cell-seeded scaffold granules. After 4 h of incubation at 37 ◦C, absorbance of conditioned medium was measured at 570 and 600 nm by means of Tecan Plate reader (Tecan, Crailsheim, Germany) and the percentage reduction of alamarBlue was calculated according to the manufacturer's instructions. Three samples were analyzed for each group and the mean value and standard deviation were calculated.

#### *4.6. hMSCs Distribution on Scaffold Granules*

To visualize hMSCs, seeded on β-TCP scaffold, before and after freezing-thawing procedure, Calcein AM (BD Pharmingen, Heidelberg, Germany) and DAPI (Life Technologies, Darmstadt, Germany) staining were applied. Scaffold granules with cells were transferred into a well of 24-well plate and 1 mL of pre-warmed growth medium with 20 μM Calcein AM was added. Cells were incubated for 40 min at 37 ◦C; washed several times with PBS; and DAPI (1 μg/mL in PBS) was added. After 10 min of incubation, cells were washed several times with PBS. hMSCs adherence and distribution on a scaffold was then assessed by directly viewing the samples with fluorescence microscopy using a Zeiss Axioobserver Z1 (Zeiss, Göttingen, Germany).

#### *4.7. Osteogenic Differentiation*

In order to evaluate the effect of cryopreservation on hMSCs osteogenic differentiation, cells after thawing were cultured for 24 h in growth medium and then growth medium was supplemented with 10-7 M of dexamethasone, 10 mM of β-glycerophosphate, and 0.05 mM of ascorbic acid-2-phosphate (Osteogenic medium, OM), all obtained from Sigma-Aldrich (Heidelberg, Germany). Cells from the control group were cultured in growth medium for 48 h after seeding, whereupon medium was changed to osteogenic-supplemented medium. Cells from both groups were cultured for another two weeks in osteogenic-supplemented medium and medium was changed every 3–4 days.

#### *4.8. Gene Expression Analysis*

For gene expression analysis, total RNA from cells was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Purity and quantity of RNA were measured using an Infinite 200PRO NanoQuant device (Tecan, München, Germany). DNase-treated RNA samples were reverse-transcribed using iScript Select cDNA Synthesis Kit (Bio-Rad, Feldkirchen, Germany) according to the manufacturer's instructions. The RT-qPCR reaction was performed using the cDNA equivalent of 10 ng RNA and the RT2 SYBR Green qPCR Mastermix (Qiagen, Hilden, Germany). All samples were amplified in duplicates using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Feldkirchen, Germany) with human gene specific primers (RT2 qPCR Primer Assays, Qiagen, Hilden, Germany), and thermal profile of 1 cycle with 10 min of 95 ◦C, 40 cycles with 15 s of 95 ◦C, and 1 min of 60 ◦C followed by dissociation curve. GAPDH was used as reference gene in each experiment. A melting curve analysis was applied to ensure the specificity of the PCR procedure. Relative quantification of messenger RNA (mRNA) levels of the target genes was analyzed using the comparative CT (threshold cycle values) method (2−<sup>Δ</sup>Ct) [38]. The results are presented as relative quantification (RQ), which is expression fold change compared to the reference gene. Three samples were analyzed for each group and mean value and standard deviation were calculated for further analysis.

#### *4.9. Alkaline Phosphatase Expression Assay*

As a marker of osteogenesis, ALP expression was assessed on day 7 of osteogenic culture according to the manufacturer's protocol (Sensolyte pNPP Alkaline Phosphatase Detection Kit, Anaspec Inc., Köln, Germany). Granules with seeded cells were washed twice with 1x assay buffer and cells were lysed with lysis buffer (0.02% Triton X-100 in 1× assay buffer). Cell lysates were collected into fresh micro tubes and processed for ALP activity measurements. Absorbance was measured at 405 nm by means of Infinite 200PRO NanoQuant plate reader (Tecan, Crailsheim, Germany). The absolute ALP value for each sample was calculated against an alkaline phosphatase standard curve.

#### *4.10. ELISA*

The effect of cryopreservation on interleukin (IL)-6 and IL-10 expression in hMSCs was evaluated using ELISA. The concentration of interleukins was measured in cell conditioned medium collected at days 1, 2, 3, and 7 post-thawing by means of human IL-6 DuoSet ® ELISA and human IL-10 DuoSet ® ELISA (R&D Systems, Wiesbaden-Nordenstadt, Germany) in accordance with the manufacturer's instructions. The absorbance was measured at 450 nm and cytokine concentrations were calculated based on calibration curve plotted with different standards concentrations by means of Magellan Software.

#### *4.11. Statistical Analysis*

All experiments were done in triplicates. The data are presented as mean ± SD and significance level was set at *p* < 0.05. Nonparametric Kruskal–Wallis test followed by Bonferroni-corrected multiple Conover–Iman post hoc analysis was consequently applied. A *p* < 0.05 indicated statistical significance. Statistics were calculated using the software Bias 11.12 (Epsilon-Verlag, Darmstadt, Germany).

#### **5. Conclusions**

Our findings show that hMSCs can be freeze-thawed on β-TCP scaffold through a technically simple procedure and thus provide a critical step towards the development of clinical-grade BTEs for functional bone augmentation. Cells treated in this fashion sustained their metabolic activity and showed favorable osteogenic potential after cryopreservation. The detailed mechanisms as to how the cryopreservation procedure beneficially affects the osteogenic potential of hMSCs, specifically the roles of YBX1 and HIF1α genes, should be investigated in future studies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms23031425/s1.

**Author Contributions:** Conceptualization, P.B., H.B. and D.H.; Data curation, L.L. and A.G.; Formal analysis, L.L. and A.G.; Funding acquisition, D.H.; Investigation, L.L. and A.G.; Methodology, L.L., Z.K., S.K., H.B., I.M. and D.H.; Resources, Z.K. and S.K.; Supervision, P.B., H.B., I.M. and D.H.; Visualization, L.L. and A.G.; Writing—original draft, L.L.; Writing—review and editing, L.L., A.G., Z.K., S.K., P.B., H.B., I.M. and D.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Federal Republic of Germany/Federal Ministry for Economic Affairs and Energy on the basis of a resolution of the German Bundestag (03THW10L27).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to acknowledge Kathrin Rottluff and Alexander Schaible for technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Communication* **Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells**

**Esther Marhuenda 1,2,†, Alvaro Villarino 1,†, Maria Leonor Narciso 1,3, Marta Camprubí-Rimblas 2,4, Ramon Farré 1,2,5, Núria Gavara 1,2,3, Antonio Artigas 2,4, Isaac Almendros 1,2,5 and Jorge Otero 1,2,3,\***


**Abstract:** One of the main limitations of in vitro studies on lung diseases is the difficulty of maintaining the type II phenotype of alveolar epithelial cells in culture. This fact has previously been related to the translocation of the mechanosensing Yes-associated protein (YAP) to the nuclei and Rho signaling pathway. In this work, we aimed to culture and subculture primary alveolar type II cells on extracellular matrix lung-derived hydrogels to assess their suitability for phenotype maintenance. Cells cultured on lung hydrogels formed monolayers and maintained type II phenotype for a longer time as compared with those conventionally cultured. Interestingly, cells successfully grew when they were subsequently cultured on a dish. Moreover, cells cultured on a plate showed the active form of the YAP protein and the formation of stress fibers and focal adhesions. The results of chemically inhibiting the Rho pathway strongly suggest that this is one of the mechanisms by which the hydrogel promotes type II phenotype maintenance. These results regarding protein expression strongly suggest that the chemical and biophysical properties of the hydrogel have a considerable impact on the transition from ATII to ATI phenotypes. In conclusion, culturing primary alveolar epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and thus help in the research on lung diseases.

**Keywords:** extracellular matrix; hydrogels; alveolar cells; type II phenotype; YAP

#### **1. Introduction**

The fundamental questions on the precise mechanisms underlying alveolar epithelial cells (AECs) damage and epithelium repair in relevant diseases, such as acute respiratory distress syndrome or chronic obstructive pulmonary disease, are still unsolved. Although it is well-known that the alveolar epithelium is repaired by the proliferation of type II AECs (ATIIs), which differentiate into type I phenotype cells (ATIs), the involved mechanisms are still poorly understood [1]. In fact, a limitation hampering translational studies in lung diseases is the difficulty of maintaining the type II phenotype of primary AECs in vitro. Indeed, it is widely known that ATII-to-ATI transdifferentiation occurs very quickly in vitro; thus, primary type II AECs neither adequately proliferate, nor can be subcultured under conventional culture conditions [2].

To extend the maintenance of the type II AECs proliferative phenotype in vitro, it was proposed to coat the culture plates with hydrogels, such as Matrigel, resembling the

**Citation:** Marhuenda, E.; Villarino, A.; Narciso, M.L.; Camprubí-Rimblas, M.; Farré, R.; Gavara, N.; Artigas, A.; Almendros, I.; Otero, J. Lung Extracellular Matrix Hydrogels Enhance Preservation of Type II Phenotype in Primary Alveolar Epithelial Cells. *Int. J. Mol. Sci.* **2022**, *23*, 4888. https://doi.org/10.3390/ ijms23094888

Academic Editors: Athanasios G. Papavassiliou, Patrick C. Baer and Ralf Schubert

Received: 27 March 2022 Accepted: 26 April 2022 Published: 28 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

extracellular matrix (ECM) [3]. Moreover, pioneering studies on subculturing strategies for primary ATII cells [4] employed inhibitors of Rho kinases, as the connection between the activation of the Rho pathway and cell mechanosensing of the ECM is well-established [5]. However, although the relationship between ATII phenotype maintenance in vitro and culturing cells on ECM-like substrates was proposed three decades ago [6], the problem remains open, probably because no hydrogels derived from lung ECM were available.

Interestingly, based on an initial report for obtaining hydrogels from the ECM of decellularized lungs [7], we have recently described a procedure for preparing such hydrogels by exclusively using lung ECM, with no need to add potentially toxic external cross-linkers [8]. If used as a culture substrate, this hydrogel, which realistically mimics the native lung ECM, could be particularly well-suited for providing a physiomimetic microenvironment to primary AECs. Therefore, we hypothesized that a lung ECM-based hydrogel would slow down the ATII-to-ATI transdifferentiation mediated by the inhibition of the Rho pathway, resulting in a decrease in F-actin polymerization and the formation of focal adhesions, as well as nuclear YAP activation [9–11].

#### **2. Results**

#### *2.1. Primary Alveolar Epithelial Cells Form Monolayers on Lung-Derived Hydrogels*

Isolated AECs were grown either in lung-derived hydrogels or plates for four days. Cells were able to form a monolayer on a lung-derived hydrogel, as shown in bright-field images in Figure 1. Differences in the morphology of the cells as a function of the substrate were also noticeable as shown by confocal images in Figure 2. Throughout the culture, cells on plates started to show a more flattened shape as well as larger cytoplasms (cells cultured on plates presented, in general, sizes about 5 times larger than cells cultured on hydrogels), and the presence of vacuoles was noticeable. On the contrary, AECs cultured on hydrogels showed cuboidal shapes, and even their microvilli, a phenotypical characteristic of ATII cells [12], could be distinguished. Cells cultured on plates formed monolayers faster (day 3 vs. day 4).

**Figure 1.** Rat primary alveolar cells were cultured either on porcine lung-derived hydrogel (**A**) or on a plate (**B**). Bright field images were taken every 24 h from day 1.

**Figure 2.** Three-dimensional representation of primary alveolar type II cells cultured for three days on hydrogel (**A**) and on a plate (**B**). Nuclei are stained in blue and actin cytoskeleton in red. In the case of the hydrogel-cultured cell, it can be distinguished by a cuboidal morphology and the presence of microvilli, indicated by arrows. In the case of the cell cultured on a plate, a more spread out and wider cytoplasm can be observed showing increased size.

#### *2.2. Culture of Alveolar Epithelial Cells on Lung-Derived Hydrogels Preserves the Expression of Type II Markers for Longer Periods*

The results of the genetic expression of ATI and ATII typical markers (from day 2 to 5) are shown in Figure 3A. The gene expression of *sftpc* and *sftpb* decreased over time. There was a significant increase in ATII markers (surfactant proteins B and C) at earlier times (day 2 and day 3) in cells cultured on lung-derived hydrogels. There was no increase in ATI markers over time in cells cultured on hydrogels, in contrast to cells cultured on plates, where the expression of *pdpn* and *aqp5* increased with time (classical ATI markers). These results show that the ATII phenotype and gene expression levels were maintained for longer in lung-derived hydrogels. The results from immunostaining are shown in Figure 3B–E, revealing that the differential expression of surfactant protein C caused by the substrate was noticeable not only at the gene expression level, but also at the protein level. SPC expression was higher in cells cultured on lung hydrogels compared to that of cells cultured on a plate, as shown by the immunostainings. Furthermore, culture time affected cells differently depending on the substrate they were cultured on. Indeed, cells cultured on lung hydrogel were able to maintain the SPC expression over time, whereas cells cultured on a plate rapidly lost this ability.

**Figure 3.** ATII cells were cultured either on hydrogel (HG) or a tissue culture plate (TCP). ATII typical marker surfactant proteins C (*sftpc*) and B (*sftpb*) and ATI typical marker aquaporin 5 (*aqp5*), podoplanin (*pdpn*) were studied at different time points (from day 2 to day 5) by qPCR (**A**). Relative gene expression is shown. Expression of surfactant protein C (SPC) was studied using immunofluorescence on ATII cells cultured on hydrogel at day 2 (**B**) and day 4 (**D**) and on a plate at day 2 (**C**) and day 4 (**E**). \* *p* < 0.05, \*\*\* *p* < 0.001.

#### *2.3. Culture of Primary Alveolar Epithelial Cells in Lung-Derived Hydrogels Inhibits Type II-to-Type I Transdifferentiation by Altering the Hippo/Rho Pathway*

Since YAP is a key mechanotransduction protein, its expression was studied in cells cultured on hydrogels or plates for three days (Figure 4A,B). In cells cultured on hydrogels, the YAP protein was located mainly in the cytoplasm, which indicates that it was being phosphorylated and subsequently degraded. In contrast, in cells cultured on plates, nuclear active YAP was observed. Specifically, there was a two-fold increase in the amount of nuclear YAP when cells were cultured on a plate compared to that of cells cultured on hydrogels, indicating a higher transcriptional activity in the first group (Figure 4C).

**Figure 4.** Alveolar type II cells were cultured on hydrogel (**A**) or on a plate (**B**) and stained for YAP protein (green). YAP nuclear and cytoplasmic expressions were quantified and expressed as the nuclei/cytoplasm fluorescence ratio in both conditions hydrogel (HG) and tissue culture plate (TCP) (**C**), \*\*\* *p* < 0.001.

Owing to the role of focal adhesions (FAs) and the actin cytoskeleton in sensing extracellular matrix cues and transmitting them to the cell, the expressions of actin and paxillin, which is one of the proteins comprising FAs, were studied. Both were reported to be implicated in the hippo pathway, by inhibiting it and promoting the YAP nuclear expression. In cells cultured for three days on lung-derived hydrogels, a poor focal adhesion assembly was observed by the paxillin immunostaining. Moreover, no stress fibers were formed as indicated by the phalloidin staining (Figure 5A). On the contrary, in cells that were cultured for three days on a plate, assembled paxillin and stress fibers were clearly observed (Figure 5B). The role of Rho, which is involved in the maturation of focal adhesions and YAP regulation [13,14] was studied by the use of the ROCK inhibitor (Y27632). The results show that it could play a role in the maintenance of the ATII phenotype, as reflected by an increase in SPC in conventional culture (*p* = 0.02) (Figure 5D) together with a decrease in focal adhesion size (*p* = 0.001) (Figure 5D), suggesting that the use of HGs as a substrate for AECs culture could be inhibiting the Rho pathway, and thus allowing for type II phenotype maintenance.

**Figure 5.** Representative images of focal adhesions (FAs) in cells cultured on hydrogels (**A**) and plate (**B**). Red: phalloidin, green: paxillin, blue: nuclei. Quantification of the FA length (**C**) and the intensity of surfactant protein C (SPC) of alveolar type II cultured on hydrogels for 3 days with (Y27632) and without (CTRL) the addition of the ROCK inhibitor (**D**), \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### *2.4. Primary Alveolar Epithelial Cells Cultured on Lung-Derived Hydrogels Can Be Subcultured*

The potential ability to subculture primary ATII cells was studied. Cells were cultured for 3 days on lung hydrogels and, after that time, lung-derived hydrogels were digested with collagenase, and cells were seeded again on plates. Sixteen hours later, they were immunostained for typical epithelial (EpCAM) and ATII (SFTPC) markers (Figure 6). Subcultured cells were positive for both, EpCAM and SFTPC markers. These results indicate that lung-derived hydrogels allow for primary AECs subculture.

**Figure 6.** Alveolar type II cells were cultured on hydrogels for three days. After that time, they were subcultured on plates and stained for surfactant protein C (red) and EpCAM (green).

#### **3. Discussion**

We have provided evidence that using lung-derived ECM hydrogels as a culture substrate allows the maintenance of the type II phenotype in primary AECs to be enhanced. Moreover, our results for YAP and when using ROCK inhibitors on cells cultured on lung hydrogels, which preserve several proteins from their native organs [7], suggest that the biophysical properties of the hydrogel have a high impact on cell mechanosensing pathways, thereby playing a role in the maintenance of type II phenotype. This first-time study culturing cells on lung ECM-derived hydrogels, which was carried out in rat primary AECs as a proof of concept, opens the door for further research in primary human AECs, with a potential future impact on cell therapies for diseases such as pulmonary fibrosis and acute lung injury [15,16].

Similar results to those obtained in the present study were provided by Shannon and coworkers [6], who conducted the first study showing the importance of the protein content of the substrate in the maintenance of ATII morphology and phenotype. The main limitation of that first study was the inability of the cells to form a monolayer, which is easily accomplished in our lung-derived hydrogel. Since then, efforts have been made to improve the ability to maintain the ATII phenotype, which has only been achieved by media supplementation [3,17–19]. Both the biochemical and physical properties of the substrate are involved in cell behavior. It is worth noting that cells formed a monolayer slower in hydrogels than on plates, which could be due to the fact that cells proliferate quicker on the plate or that the ATII-to-ATI transdifferentiation allows for a faster substrate coverage, as ATI cells are much larger in size. In view of the differing stiffness of the hydrogels when compared to the culture plate and the cuboidal morphology of the cells, differences in their cytoskeleton distribution and the formation of focal adhesions, which are key points in the crosstalk of cell-ECM, were expected. Cells cultured on hydrogels showed shorter focal adhesions that correlated with a decreased YAP translocation to the nuclei. YAP is an important transcription factor that is implied in the regulation of several genes, and it

is reported to be associated with inflammatory and epithelial damage situations in vivo where the ATII-to-ATI transdifferentiation is required [20,21]. In this case, YAP nuclear location of cells cultured on a plate could be triggering the differentiation in the ATI cell population. As Rho is implicated in the maturation of focal adhesions [14], and previous authors have pointed to it as a key factor in the transdifferentiation process [4,22], its role was studied. The determination of the individual contribution of different Rho proteins would be highly interesting. However, as it would be complex [14], we studied this effect through its inhibition using Y27632. The obtained results support the implication of Rho in the ATII phenotype, as its inhibition produced an increase in SPC and decrease in FAs only in cells cultured on a plate. The origin of this inhibition of the Rho pathway, although out of the scope of the present work, is probably related to the complex molecular composition of lung-derived hydrogels and their biomechanical properties.

Primary ATII cells have previously been subcultured [4] by coculturing them with fibroblasts as feeder cells and with the addition of the Rho inhibitors. The role of fibroblasts in maintaining the ATII phenotype is not exactly known but it could be that they work as a source of the keratinocyte growth factor (KGF). The addition of KGF has been used by other authors to increase the phenotypic features of ATII cells [3,19], and in turn, to inhibit Rho kinases, which are shown to play a key role in the ATII-to-ATI transdifferentiation [22]. In our case, the use of lung-derived hydrogels as substrates for culturing allows these cells to be subcultured without the of use of additional chemicals or cocultures. Therefore, culturing primary alveolar epithelial cells on lung ECM-derived hydrogels may facilitate the prolonged culturing of these cells, and thus help in the research of lung diseases. However, more research should be carried out regarding the number of possible passages, especially considering that factors different from the ATI-to-ATII transdifferentiation would limit the time that these primary cells could be maintained in vitro. Further research is also needed to determine which components of ECM-derived hydrogels have more impact on the maintenance of the type II phenotype in vitro. As the extracellular matrix used to produce hydrogels in the present work was obtained from lung tissue, it is expected that some key biochemical and biophysical factors specific to the lung are being preserved in the process of hydrogel production. The identification of these factors, although out of the scope of the present work, would allow the development of novel optimized scaffolds for the in vitro culture of pulmonary cells.

#### **4. Materials and Methods**

Unless otherwise specified, all reagents were purchased from ThermoFisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (Saint Louis, MO, USA).

#### *4.1. Decellularized Lung Extracellular Matrix Hydrogels Preparation*

Lung-derived hydrogels were prepared by following a previously described protocol [23]. Briefly, porcine lungs were decellularized by consecutive perfusion of the following reagents through the vasculature and the airways: 0.1% Triton X-100 and 2% SDC for 24 h at 4 ◦C, and NaCl 1 M and DNase solution for 1 h at 4 ◦C. Three washes of miliQ water were performed between consecutive reagent perfusion, and a last wash of PBS 1X was carried out. Decellularized lungs were cut into small pieces, freeze-dried (Telstar Lyoquest-55 Plus, Terrassa, Spain) and milled in liquid N2 (SPEX SamplePrep, Metuchen, NJ, USA). The obtained powder was resuspended at 20 mg/mL in 0.01 M HCl and pepsin digested at a 1/10 proportion under magnetic stirring at room temperature for 16 h. To produce hydrogels, the digested solution was pH-adjusted to 7.4 ± 0.4 by using 0.1 M NaOH and incubated at 37 ◦C for 20 min.

#### *4.2. Primary Alveolar Epithelial Cells Isolation*

The procedure was approved by the Ethical Board for Animal Research of the University of Barcelona, in compliance with regional, national and European regulations. Rat lungs were obtained from 180–250 g Sprague Dawley male rats. The animals were intraperitoneally anesthetized with 1 g/kg urethane and euthanized by exsanguination. AECs were isolated following a protocol described in [24]. Briefly, lungs were perfused with saline through the pulmonary artery and were resected en bloc. Three bronchioalveolar lavages were performed with 10 mL of PBS 1X to remove alveolar macrophages. Then, lungs were digested with 50 mL of 0.25% of trypsin through the airways for 30 min, cut into small pieces, and digested in a 100 units/mL DNase solution. The resulting suspension was filtered through a 100 μm and a 40 μm mesh and centrifuged through a Percoll (GE HealthCare, Chicago, IL, USA) gradient at 500× *g* for 20 min. The band containing ATII cells was recovered and digested using 20 units/mL of DNase. The resulting solution was centrifuged for 15 min at 500× *g*, and the pellet was resuspended in DCCM-1 (Biological Industries, Kibbutz Beit Haemek, Israel) medium and cultured for 1 h. Subsequently, medium containing ATII non-adherent cells was recovered and centrifuged for 10 min at 800× *g*. Cells were finally counted and seeded either on 24-well culture plates or on top of lung-ECM hydrogels.

#### *4.3. Rho Kinase Inhibition Assay*

For the inhibition of the Rho pathway, Y27638 (10 μM) was added to the medium for 24 h.

#### *4.4. Reverse Transcription qPCR*

For studying the expression of ATI and ATII markers, cells were cultured either on plastic or lung-derived hydrogels for different times, and RNA was subsequently extracted from samples by employing the RNeasy kit (Qiagen, Hilden, Germany). The cDNA was obtained by a reverse transcription-polymerase chain reaction (TaqMan Reverse Transcription Reagents, Invitrogen, Waltman, MA, USA) according to the manufacturer's instructions. The expression level of surfactant protein C (*sftpc*), surfactant protein B (*sftpb*), aquaporin 5 (*aqp5*) and podoplanin (*pdpn*) was studied using the Taqman Fast Advanced Master Mix and the TaqMan Gene Expression Assays in a StepOnePlus thermocycler (Applied Biosystems, Waltham, MA, USA). The expression level of genes was normalized to the constitutively expressed gene PPIA and calculated using the 2−ΔΔCt method [25].

#### *4.5. Immunohistochemistry and Image Processing*

For immunohistochemistry experiments, cells were fixed with 4% paraformaldehyde for 30 min. Primary antibodies were incubated overnight, and secondary antibodies were incubated for 2 h at 37 ◦C. Nuclei were stained with Hoechst 33342. To avoid unspecific binding, especially in the hydrogel samples, a blocking buffer consisting of 2% BSA (Thermo Fisher, MA, USA) diluted in PBS 1X (Gibco, MA, USA) was employed for 40 min. Primary antibodies employed were rabbit anti-SFTPC (Invitrogen, Waltman, MA, USA), mouse anti-YAP (Santa Cruz Biotechnology, Dallas, TX, USA) and EpCAM (Miltenyi, Bergisch Gladbach, Germany). Secondary antibodies used were goat anti-rabbit cy5 (Abcam, Cambridge, UK) and goat anti-mouse Alexa Fluor 488 (Abcam, Cambridge, UK). Images were acquired with a Nikon Confocal Eclipse Ti microscope using a 20 × Plan Fluor Multi-immersion objective (0.75 NA) in the case of the SFTPC, EpCAM and YAP staining with a 10× objective (0.3 NA) for the bright field images, and a 100× objective (1.45 NA) for the paxillin and actin images. Nuclear images were obtained at 450 nm when illuminating the sample at 408 nm. Samples were excited at 488 nm and acquired at 515 nm for YAP, EpCAM and Paxillin images, and excited at 543.5 nm and acquired at 605 nm for SFTPC and actin stains.

For the analysis of YAP images, five images per condition were randomly selected and analyzed using a blind procedure with ImageJ Software. Quantification of the ratio nuclear fluorescence/cytoplasmic fluorescence was assessed following a previously described procedure [26] with slight modifications. To calculate the total cell fluorescence, a triangle threshold was employed, and the integrated fluorescence was calculated in the YAP channel. For calculating the YAP nuclear fluorescence, the perimeter of the nuclei was delimited by

the Huang threshold in the DAPI channel. After that, the resulting mask was redirected to the YAP channel and the integrated intensity contained in the nuclear perimeter was obtained. To calculate the cytoplasmic fluorescence, the differences in the intensities in the total cell and in the nuclei were calculated.

For focal adhesion length measurements, five representative adhesions per cell in paxillin stainings at the cell edge were manually quantified with Image J Software.

#### *4.6. Statistical Analysis*

Data are expressed as mean ± SE unless stated otherwise. Statistical analysis was performed with Graphpad Prism software. Differences in ATI/ATII markers gene expression and YAP nuclear/cytoplasmic expression were analyzed using paired *t*-test. A *p*-value < 0.05 was considered significant.

**Author Contributions:** Conceptualization, E.M., I.A. and J.O.; Formal analysis, E.M., A.V., A.A., R.F., I.A. and J.O.; Funding acquisition, R.F., I.A., N.G. and J.O..; Investigation, E.M., A.V., I.A. and J.O.; Methodology, E.M., A.V., M.L.N., M.C.-R., I.A. and J.O.; Project administration, R.F., I.A. and J.O.; Resources, N.G., A.A., R.F., N.G., I.A. and J.O.; Software, E.M., M.L.N. and N.G.; Supervision, I.A. and J.O.; Writing of original draft, E.M. and J.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Science, Innovation and Universities, grants numbers SAF2017-85574-R, DPI2017-83721-P, PID2019-108958RB-I00, and PGC2018-097323-A-I00, and by European Union's Horizon 2020, Marie Sklodowska-Curie, grant 821772.

**Institutional Review Board Statement:** Animal experiments were approved by the Ethical Committee for Animal Research of the University of Barcelona.

**Data Availability Statement:** Data supporting the findings of this study are available from the corresponding authors upon reasonable request.

**Acknowledgments:** The authors wish to thank Miguel A. Rodríguez from the Unit of Biophysics and Bioengineering for his excellent technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

