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Surface Tension Guided Hanging-Drop: Producing Controllable 3D Spheroid of High-Passaged Human Dermal Papilla Cells and Forming Inductive Microtissues For Hair-follicle Regeneration Bojie Lin, Yong Miao, Jin Wang, Zhexiang Fan, Lijuan Du, Yongsheng Su, Bingcheng Liu, Zhiqi Hu, and Malcolm M.Q. Xing ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00202 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016

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ACS Applied Materials & Interfaces

Surface Tension Guided Hanging-Drop: Producing Controllable 3D Spheroid of High-Passaged Human Dermal Papilla Cells and Forming Inductive Microtissues For Hair-follicle Regeneration

Bojie Lina,b,c, Yong Miaoa, Jin Wanga, Zhexiang Fana, Lijuan Dua, Yongsheng Sua , Bingcheng Liua, Zhiqi Hua,* , and Malcolm Xingb,c,* a

Department of Plastic and Aesthetic Surgery, Nanfang Hospital of Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, Guangdong 510515, China b Department of Mechanical Engineering and Department of Biomedical & Medical Genetics, University of Manitoba, 75A Chancellors Circle, Winnipeg, Manitoba R3T 2N2, Canada c Children’s Hospital Research Institute of Manitoba, 715 McDermot Ave, Winnipeg, Manitoba R3E 3P4, Canada

*Address correspondence to: [email protected] (mx) [email protected] (hz)

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Abstract Human dermal papilla (DP) cells have been studied extensively when grown in the conventional monolayer. However, due to great deviation from the real in vivo three-dimensional (3D) environment, these two-dimensional (2D) grown cells tend to lose the hair-inducible capability during passaging. Hence, these 2D caused concerns have motivated the development of novel 3D culture techniques to produce cellular microtissueswith suitable mimics. The hanging-drop approach is based on surface tension-based technique and the interaction between surface tension and gravity field which makes a convergence of liquid drops. This study used this technique in a converged drop to form cellular spheroids of dermal papilla cells. It leads to a controllable 3Dspheroid model for scalable fabrication of inductive DP microtissues. The optimal conditions for culturing high-passaged (P8) DP spheroids were determined firstly. Then, the morphological, histological and functional studies were performed. In addition, expressions of hair-inductive markers including alkaline phosphatase, α-smooth muscle actin and neural cell adhesion molecule were also analyzed by quantitative RT-PCR, immunostaining, and immunoblotting. Finally, P8-DP microtissues were co-implanted with newborn mouse epidermal cells (EPCs) into nude mice. Our results indicated that the formation of 3D microtissues not only endowed P8-DP microtissues many similarities to primary DP, but also confer these microtissues an enhanced ability to induce hair-follicle (HF) neogenesis in vivo. This model provides a potential to elucidate the native biology of human DP, and also shows the promising for the controllable and scalable production of inductive DP cells applied in future follicle regeneration.

Key words: surface tension; hanging-drop; 3D culture; dermal papilla; hair-follicle induction.


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Introduction Hair loss is a common disorder that affects mankind due to reasons such as aging, diseases and medications. Although being not life-threatening, baldness has influence not only on social interaction but also psychological condition1-2. Current therapeutic options include either medication or autologous hair transplantation. However, both treatments are only effective for mild alopecia and still remained challenged by the inability to form de novo hair follicles (HFs) in bald scalp. With the recent advances achieved in regenerative medicine and tissue engineering, re-arranging inter-follicular cells and thus bio-engineering the HF becomes a promising alternative3-5. Hair follicles (HFs) are ectodermal organs with composite three-dimensional (3D) structure that contains two major parts--epithelium and mesenchyme. The epithelium mainly consists of epidermal cells and mesenchyme has dermal components. Among this complex organization, the dermal papilla (DP) is a highly specialized mesenchymal cell population located at the base of HF, which has an indispensable role in the epithelial–mesenchymal interactions that enable normal HF morphogenesis and postnatal HF cycling6-7. It has been proved that cultured DP cells retain the ability to induce HF neogenesis in non-hair-bearing skin8-9. However, DP cells tend to lose their trichogenicability in vitro, where cells were cultured and studied under traditional two-dimensional (2D) monolayers 2, 7, 10.In living organisms, most cells are well-assembled into 3D organizations, where the cell-cell and cell-extracellular matrix (ECM) interactions that are considered to be crucial for cell function and tissue morphogenesis usually occur. Furthermore, un-natural 2D system does not reflect the real in vivo environment and may compromise the distinct features and inductive function of DP cells11-12. Therefore, to mimic an in vivo-like condition has led to an alternative strategy for in vitro culture, in which DP cells are grown into a three-dimensional (3D) microtissue13. At present, most 3D culture systems existing used scaffolds to support the growth of follicle cells, where they provided extracellular components (e.g. collagen, hydrogel, gelatin-sponge, artificial scaffold) to facilitate cell growth or aggregation5, 14-16, while a 3D condition could also been achieved by low-adhesion and micro-fluid technologies17-18. However, these culture systems often suffered from problems and limits such as tedious and time-consuming procedure for operating, producing spheroids with variable sizes, hard to handle or control, and low-throughput. Enlightened by the theory of surface tension, the hanging-drop technique can be used to generate multi-cellular microtissues. Unlike the aforementioned 3D systems, this surface tension-based fabrication has its unique advantages. First, to form spheroid microtissues requires neither additional supplements nor artificial scaffold components utilizing this system. Furthermore, such a biomimetic technique allows for target cells and tissue biology to be studied in vitro under the condition that closely simulate the in vivo situation. Third, no complicated technologies and skills are needed for handling13, 19. A traditional hanging-drop culture was usually prepared by depositing drops consisted of cell suspension onto the lid of a petri dish. When the lid is inverted, drops are hung from the bottom side with a meniscus shape due to hydrophilicity of the lid and liquid surface tension. Using the aforementioned technique, previous studies have described that the inductive phenotypes of DP cells could be partially reversed by reassembling them into 3D spheroids20, in which they could further elicit new follicle formation in recipient skin12. Nonetheless, this traditional method also possessed several limits and shortages, such as the



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vulnerability to fall or spread out, inability for long-term culture, and difficulty to scale up the production etc. Recently, a great advance of surface tension-based fabrication technique has been achieved by using the novel hanging-drop microplates, which have exhibited distinct superiorities21. On one hand, the hanging-drop arrays endows a more facile and feasible way to precisely control over the formation of well-organized microtissues, in which the multi-cellular spheroids can be produced in a uniform-sized manner 19, 22. On the other hand, the semi-microarrays can be compatible with high-throughput and automated technologies, thus providing a relatively adequate cell sources21. In this study, we sought to establish an in vitro DP model based on surface tension fabrication and explored whether this model could enable a highly controllable and stable production of inductive human high-passage DP cells.

Materials and Methods Isolation of human DP cells and 2D Cell Culture Under the approval by the Medical Ethical Committee of the Southern Medical University (Guangzhou, China), human scalp samples were collected from patients who underwent face lifting surgery after obtaining informed consent. Isolation and expansion of DP cells were performed as previously described23. Micro dissected dermal papillae were transferred onto plastic dishes and cultured in the Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY) supplemented with 1% (v/v) penicillin-streptomycin and 20% (v/v) fetal bovine serum (FBS, Gibco) in a humidified 5% CO2incubatorat 37oC.The cultivation of explants was kept for 7 days in medium, which was changed every 3 days. Once the outgrowth reached to 70% confluence, DP cells were harvested with 0.25% (w/v) trypsin/EDTA (Gibco) and then subculture was conducted with a split ratio of 1:2. Afterwards, cells were maintained in the DMEM supplemented with 10% FBS. DP cells at passage 8 were used in the following experiments. Establishment of DP microtissues in 3D hanging drop cultures For 3D cell cultures, Perfecta3D® Hanging Drop Plates (3D BiomatrixInc., Michigan, USA) were used. The hanging-drop culture system was prepared according to the manufacturer’s protocol. To test the effects of seeding density on DP microtissue formation, various cell densities (0.10, 0.25, 0.5, and 1×104cells/40µl/well) were seeded into96-well Hanging drop plates. Then, the size distributions of DP microtissues were summarized to determine the optimal condition for culture. All DP cells were cultured in complete medium (DMEM+10% FBS), which was changed every 2daysusing the multi-channel pipette. Morphology and diameter of DP spheroids were recorded under a reverse phase-contrast microscope (IX61 FL, Olympus, Japan) for up to 7 days. The following experiments were then conducted under the determined optimal condition. Dynamic imaging, morphological, histological and functional characterization of DP microtissues For dynamic observation of DP spheroid formation, DP cells were seeded into 96-well hanging drop microplates and then recorded using phase-contrast microscope. Recording time ranged from day0 to day7 after seeding. To confirm the cell viability, DP spheroids on day 7 of culture were stained with LIVE/DEAD Viability/Cytotoxicity Kit (L3224, Invitrogen Co.),incubated for 20 min at 37°C,and further imaged within the plates by fluorescence microscopy (IX61 FL, Olympus, Japan).


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For scanning electron microscopy (SEM), both P8-DP cells and DP spheroids at day 7 of cultivation were harvested and then fixed in the 0.1M Sorensen’s buffer that contains2.5% glutaraldehyde for about 2 h. Next, the samples were rinsed with 0.1 M Sorensen’s buffer containing5% sucrose for three cycles of washing (5 min at each time). After being dehydrated with graded alcohol and then drying, the samples were further sprayed with gold prior to observation by SEM (JEOL 5900,Japan). For histological examination, DP microtissues were collected in a 1.5-ml tube, fixed with 4% paraformaldehyde in PBS for 15 min, washed, paraffin-sectioned and then stained with haematoxylin and eosin (H&E).For immunohistochemistry, DP microtissues were fixed and further stained with proliferation marker Ki67 (anti-Ki67 antibody, Abcam) and TUNEL reaction mix (In Situ Cell Death Detection Kit, AP., Roche,Germany) according to the manufacturer’s protocol. Images were recorded using a fluorescence microscopy. Subsequently, the percentage numbers of positive cells were calculated by normalizing them with the total cell number ofDP spheroids using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA).Quantification was carried out in triplicate with three microtissues analyzed per sample. For an in vitro simulating transfer culture, DP microtissues formed at day 7 of culture were carefully removed and injected onto new culture dishes using a 200-ml pipette (4844, Corning Incorporated Life Sciences, USA) for another 7 days of incubation. Cell morphology and outgrowth from microtissues were observed under the reverse phase-contrast microscope or SEM. Primary DP was used as a positive control. Quantitative real time-polymerase chain reaction Total RNA was extracted from P8-DP microtissues, adherent P8-DP cells, and dermal-fibroblast spheroids using an RNAiso Plus reagent (Takara, Dalian, China). cDNA was synthesized from 2mg of total RNA with a SYBR PrimeScriot RT-PCR Kit (Takara). Quantitative RT-PCR (qRT-PCR) was carried out using a SYBR PrimeScriot RT-PCR Kit (Takara) on a Stratagene MX3005P QRT-PCR system (Agilent Technologies, Santa Clara, CA). All the aforementioned steps were conducted according to the manufacturer’s instruction. Fold change of each target gene has been normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The primers used in this study are listed below: ALP 5’-ATTGACCACGGGCACCAT-3’ and 5’-CTCCACCGCCTCATGCA-3’; α-SMA 5’-GCTTCCCTGAACACCACCCAGT-3’ and 5’-GCCTTACAGAGCCCAGAGCCAT-3’;NCAM 5’-TCCGAGTTCAAGACGCAGCCA-3’ and 5’-GGTGGAGACAATGGAACAGGGGT-3’; GAPDH 5’-GCACCGTCAAGGCTGAGAAC-3’ and 5’-TGGTGAAGACGCCAGTGGA-3’. Immunofluorescent staining Immunostaining were performed according to routines. Briefly, DP samples were collected from 3D hanging drop plates, washed, fixed in 4% paraformaldehyde and followed by paraffin-embedded. The following rabbit monoclonal primary antibodies were used: ALP (Epitomics, Burlingame, CA),α-SMA (Epitomics, Burlingame, CA), and NCAM (Epitomics, Burlingame, CA).On the following day, samples were incubated with the corresponding mouse anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA). All staining procedures were conducted according to the manufacturer’s guidelines. Images were taken utilizing a fluorescent microscope (IX61 FL, Olympus, Japan). Western blot analysis Total cell lysates were collected and prepared from DP microtissues. Then 30μg of protein for



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each sample was loaded per lane and subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. Primary antibodies were incubated as follows: anti-ALP monoclonal antibody, 1:1000; anti-NCAM monoclonal antibody, 1:5000; anti-α-SMA monoclonal antibody, 1:1000; and anti-GAPDH monoclonal antibody, 1:1000 (Santa Cruz Biotechnology, Inc.,SantaCruz, CA, USA). Finally, immune complexes were detected by an enhanced chemiluminescence (ECL) kit (Santa Cruz). Results were further quantified by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA). In vivo HF induction of DP microtissues To determine the hair-inductivity of DP microtissues, an established patch assay was performed24. Briefly, freshly isolated epidermal cells were obtained from newborn mice (C57BL/6) epidermis as previously described25. Next, after 7 days of culture, a total of 60 P8-DP spheroids harvested from hanging drop array plates were combined with 1×106 epidermal cells and then subcutaneously implanted into the dorsal side of 7-9 weeks old nude mice (BALB/cAJcl-nu). In some experiments, P8-DP cells were labeled with the fluorescent dye CM-Dil (Invitrogen, Carlsbad, CA) for in vivo cell-tracking26 prior to microtissue fabrication. Either newborn mouse epidermal cells alone (1×106 cells) or P8-DP 2D cells mixed with epidermal cells (1×106 cells) were used as the control of experiment. Animals were purchased from the Experimental Animal Centre in Southern Medical University (Guangzhou, China).All experiments that approved by the Institutional Animal Care and Use Committee were carried out in triplicate. After 5weeks of implantation, all animals were sacrificed. For macroscopic observation, dissected samples were first characterized and imaged under a stereoscope. For further histological examination, the specimens were fixed in 10% paraformaldehyde buffered solution, paraffin-sectioned and processed for hematoxylin and eosin (H&E) staining. Frozen sections of grafts were also prepared. Induced HFs were observed and imaged under alight phase-contrast or fluorescent microscope. Statistical analysis All experiments were conducted in 96-well Hanging Drop array plates. All data are expressed as the mean±SD from three independent experiments. All statistical analysis was done with SPSS statistical software, version 17.0. The independent samples t-test was performed for comparison of microtissues among groups. Differences between qRT-PCR or western blot results were evaluated by one-way ANOVA. A p-value of

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