Reconstruction of the lymphatic system by transplantation of a centrifuge-based bioengineered lymphatic tissue

doi:10.21203/rs.3.rs-4394953/v1

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Reconstruction of the lymphatic system by transplantation of a centrifuge-based bioengineered lymphatic tissue

https://doi.org/10.21203/rs.3.rs-4394953/v1

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The increasing incidence of cancer and surgical procedures for its treatment have accelerated the demand for the development of treatments for secondary lymphedema after lymphadenectomy (LD). We demonstrated that a bioengineered tissue with a lymphatic network composed of lymphatic endothelial cells and mesenchymal stem cells fabricated by a centrifugal cell stacking technique effectively treated secondary lymphedema. The centrifuge-based bioengineered lymphatic tissue (CeLyT) survived long after transplantation and restored the lymphatic flow in LD mice. CeLyTs transplanted into LD mice formed a lymph node-like structure and suppressed lymphedema in LD mice. In addition, the lymph node-like structure was composed of transplant- and host-derived cells including a wide range of immune cells. Furthermore, an injection with the immunostimulant CpG1018 induced the release of proinflammatory cytokines in the lymph node-like structure formed in LD mice. Taken together, CeLyTs composed of lymphatic endothelial cells and mesenchymal stem cells reconstructed the lymph node and has great potential for the treatment of secondary lymphedema.

Biological sciences/Biotechnology/Tissue engineering

Biological sciences/Biotechnology/Regenerative medicine

Biological sciences/Biotechnology/Biomaterials/Tissues

Biological sciences/Biotechnology/Stem-cell biotechnology

Biological sciences/Biotechnology/Cell delivery

Lymphatic vessels, which are mainly composed of lymphatic endothelial cells (LECs), connect hundreds of lymphoid organs, transport tissue metabolites, and peripheral antigens [1,2]. Lymph nodes are the organs responsible for immune surveillance and immunomodulatory functions, and are connected by numerous lymphatic vessels [3]. Dysfunction of lymphatic vessels caused by congenital mutations or cancer treatment results in primary and secondary lymphedema, respectively [4-6]. Secondary lymphedema is often caused by lymphadenectomy (LD) during the surgical treatment of cancer [7,8]. Because lymph nodes do not regenerate once removed, patient quality of life is severely impaired by recurrent complications including interstitial fluid retention and limb cellulitis [9]. Liposuction [10], manual lymphatic drainage [11], and lymphatic anastomosis are treatment options for secondary lymphedema, but their edema-suppressing effect is limited [12,13]. Additionally, autologous lymph node transplantation for lymph node reconstruction has been attempted, but there is insufficient evidence of efficacy [14].

Cellular medicine is an innovative cell-based therapeutic that has demonstrated therapeutic potential for intractable diseases. For secondary lymphedema, mesenchymal stem cell (MSC) transplantation has been performed in medical institutions and has received much attention as a solution for lymphedema. However, there is no evidence of lymphatic regeneration or a therapeutic effect on lymphedema [15,16]. Recently, many attempts have been made to treat secondary lymphedema by forming lymphatic vessels using three-dimensional cellular structures [17-19]. Of these, three-dimensional cellular structures composed of LECs and fibroblasts fabricated using a cell stacking technique by coating functional proteins on the cell surface were reported to form a lymphatic network inside the structures, demonstrating the formation of a lymphatic lumen structure after transplantation in mice [20-22]. However, the cellular coating process with functional proteins is extremely complicated and their immunogenicity may be a major problem for clinical applications. Furthermore, this cellular structure has not been effective for the treatment of secondary lymphedema. Therefore, lymph node regeneration or reconstruction using therapeutic cells has not been achieved, and the development of a better therapeutic method is desired.

This study attempted to develop a bioengineered three-dimensional tissue composed of LECs and MSCs, which has immunomodulatory functions and can prolong the survival of transplants [23-25] for lymph node reconstruction. To fabricate the bioengineered tissue simply, we established a centrifugal cell stacking technique with no additives including functional proteins and complicated handling (Fig. 1). This bioengineered tissue termed “centrifuge-based bioengineered lymphatic tissue” (CeLyT) formed a lymphatic network inside the tissue during culture for several days. CeLyTs induced the formation of lymph node-like structures, with characteristics similar to lymph nodes, after transplantation into mice, and the formation of this lymph node-like structure suppressed edema in LD mice. Therefore, CeLyTs composed of LECs and MSCs might be a cell-based therapeutic strategy for secondary lymphedema.

Fabrication and characteristics of CeLyTs

To fabricate the CeLyT, we optimized the centrifugal conditions for cells after seeding on the insert of a Transwell plate by observing the cells using a fluorescence microscope. Under the evaluated centrifugation conditions (centrifugation speed from 50 ×g to 1000 ×g and centrifugation time from 10 sec to 3 min), seeded green fluorescent protein (GFP)-expressing mouse MSC C3H10T1/2 cells (mMSCs) settled down uniformly and reproducibly on the bottom of the insert of the Transwell plate at a speed of 750 ×g and a time of 30 sec (Supplementary Fig. 1a, b). In addition, these conditions stacked mMSC/GFP cells uniformly and reproducibly regardless of the number of centrifugations (Fig. 2a). Thus, we termed the centrifugation-based cell stacking process a centrifugal cell stacking technique. Then, we fabricated a three-layered bioengineered tissue composed of LECs (internal) and MSCs (external) by stacking MSCs, LECs, and MSCs in this order using the centrifugal cell stacking technique. MSCs were used because they support and prolong the survival of a transplant [23-25], and they should be located outside of the bioengineered tissue. The fabricated bioengineered tissues were cultured for five days to form a lymphatic network in the tissue with reference to a previous report [21]. Fig. 2b, c shows an image of the bioengineered tissue from the top of the well and a microscopic cross-sectional image of the tissue (hLEC+mMSC tissue and hLEC+hMSC tissue) fabricated using primary human LECs (hLECs) and mMSCs or primary human MSCs (hMSCs). These images show that hLEC+MSC tissues had a uniform sheet-like structure of 30–50 mm thickness. To examine differences between hLEC+MSC tissues prepared by the centrifugal cell stacking technique and mMSC tissues or hLEC/mMSC mixed tissues prepared by single seeding without centrifugation, a lymphatic endothelial cell marker Prox-1 was detected in these tissues by immunofluorescence staining. Especially, the expression of Prox-1 in the bioengineered tissues prepared by the centrifugal cell stacking technique was strongly detected (Fig. 2d). Of note, the mouse fibroblast cell line NIH3T3 did not form a sheet-like structure using the centrifugal cell stacking technique, and mMSCs seeded at the same cell number formed a non-uniform sheet-like structure (Fig. 2b and Supplementary Fig. 1c). Therefore, an optimal centrifugal cell stacking technique is required to produce a uniform bioengineered tissue. In addition, Z-stack images of hLEC+mMSC tissues showed that the locations of CellTracker™ Green-labeled mMSCs and CellTracker™ Orange-labeled hLECs were outside (detected at a depth of 0–35 mm from the top) and inside (detected at a depth of 10–30 mm, especially 10–20 mm from the top) in the tissue, respectively (Fig. 3a). These data demonstrated that a three-layered bioengineered tissue composed of LECs and MSCs was successfully fabricated by the centrifugal cell stacking technique. To observe the structure of the bioengineered tissues in more detail, hLEC+mMSC tissues fabricated using CellTracker™ Green-labeled mMSCs and CellTracker™ Orange-labeled hLECs were observed in two or three dimensions by fluorescence microscopy. hLECs formed a lymphatic network in the tissue as expected (Fig. 3b and Supplementary Fig. 1d, e). Centrifuge-based bioengineered lymphatic tissues composed of LECs and MSCs will be referred to as CeLyTs and represented as LEC+MSC tissues (the former cells are inside and the latter cells are outside the tissue, respectively) unless otherwise indicated.

To evaluate the survival of the internal and external cells during the culture period to form lymphatic networks, a NanoLuc luciferase (Nluc)-expressing mMSC/Nluc+mMSC tissue was prepared. The relative light units derived from live cell-releasing Nluc in the culture supernatant were almost constant throughout the culture period (Fig. 3c). A similar result was obtained for the luminescence images of a firefly luciferase (fluc)-expressing mMSC/fluc+mMSC tissue, where luminescence signals were detected throughout the tissue (Fig. 3d). In addition, the fluorescence microscopic observation of an hMSC+hMSC/carboxyfluorescein succinimidyl ester (CFSE; for live cell staining) tissue showed that the external mMSCs were also alive (Fig. 3e). These results demonstrated that the CeLyTs formed a lymphatic network by culturing for several days without inducing cell death. To evaluate the characteristics of the CeLyTs, the expressions of two lymphatic endothelial cell marker genes, Podoplanin (PDPN, also known as gp38 and T1a) and vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), were compared with those of other cells or tissues. The gene expressions in hLEC+mMSC tissue were dramatically increased compared with those in an hLEC/mMSC mixed tissue prepared by single seeding without centrifugation, and were also higher than those in monolayered hLEC/mMSC (Supplementary Fig. 2a). In addition, the gene expressions in hLEC+hMSC tissues increased with time during the culture period (Supplementary Fig. 2b). Flow cytometric analysis showed that the geometric mean fluorescence intensity of the hLEC/CFSE+hMSC tissue was significantly higher than that of the hLEC/CFSE/hMSC mixed tissue prepared by single seeding without centrifugation (Supplementary Fig. 2c), indicating the survival of internal LECs was maintained in CeLyTs. Next, we evaluated the contribution of MSCs to the network formation of LECs because MSCs expressed and produced collagen 1A2, which is important for the formation of endothelial cell vascular networks [26]. The expression of collagen 1A2 was observed throughout the mMSC sheet prepared by centrifugation and was higher than that in suspended mMSCs (Supplementary Fig. 2d-f). The shape of seeded hLECs on the mMSCs sheet changed to a thick spindle shape 5 days after seeding (Supplementary Fig. 2g).

Lymphatic structure formation after CeLyT transplantation

To confirm the usefulness of CeLyTs fabricated with MSCs for survival after transplantation, the survival periods of suspended mMSC/Nluc cells and various bioengineered tissues were evaluated by in vivo imaging. An mMSC/Nluc+NIH3T3 tissue was prepared as a control using as previously reported [20]. The luminescence signals of suspended mMSC/Nluc cells and mMSC/Nluc+NIH3T3 tissues disappeared 3–4 days after transplantation, and those of mMSC/Nluc+hMSC and hLEC+mMSC/Nluc tissues persisted for at least 21 days (Fig. 4a,b), indicating the long-term survival of internal and external cells in CeLyTs. Fig. 4c shows the higher and longer survival period of internal mMSC/Nluc cells in mMSC/Nluc+hMSC tissues compared with that of suspended mMSC/Nluc cells, indicating that the internal cells in CeLyTs survived for a long time when fabricated with MSCs. Next, we evaluated the formation of lymphatic vessels after subcutaneous transplantation of hLEC+mMSC tissues to mice. The expressions of Prox-1 and an endothelial marker CD31 were detected in skin tissues transplanted with hLEC+mMSC tissues 14 days after transplantation, although no expression of Prox-1 was detected in normal skin (Fig. 4d). In addition, the shape of LECs expressing Prox-1 and CD31 in the hLEC+mMSC tissue-transplantation group showed the formation of luminal structures by lymphatic cells, as previously reported [21].

Restoration of lymphatic flow in LD mice after CeLyT transplantation

We evaluated the therapeutic effect of CeLyTs. First, we prepared LD mice with impaired lymphatic flow, which caused lymphedema, by removing the popliteal and inguinal lymph nodes. Then, we evaluated the lymphatic flow in these mice using a lymphatic flow testing reagent indocyanine green (ICG) by in vivo imaging. ICG signals were detected in the inguinal lymph nodes of normal mice (no treatment) 1 h after ICG injection, whereas no signals were detected in the inguinal lymph nodes of ICG-injected LD mice 1, 7, or 14 days after lymph node removal (Supplementary Fig. 3a). ICG fluorescence signals in the inguinal lymph nodes (the transplantation site) in LD mice transplanted with suspended mMSC/Nluc cells or various bioengineered tissues were analyzed 1 h after ICG administration by in vivo imaging. hLEC+mMSC/Nluc tissue- and hLEC+hMSC tissue-transplanted LD mice showed fluorescence signals at the transplantation site on day 21, whereas other groups showed no signal, indicating CeLyTs restored the lymphatic flow in LD mice (Fig. 5a). To confirm the persistence of CeLyTs after transplantation into LD mice, the luciferase-derived luminescence of hLEC+mMSC/Nluc tissues was detected by in vivo imaging. Luminescence signals were detected at the transplantation site in LD mice transplanted with hLEC+mMSC/Nluc tissue at 1 and 14 days after transplantation, whereas signals in LD mice transplanted with suspended mMSC/Nluc cells were barely detectable 14 days after transplantation (Fig. 5b). In addition, hLEC+mMSC tissue formed a lymph node-like structure at the transplantation site in LD mice 21 days after transplantation (Fig. 5c and Supplementary Fig. 3b). To confirm that the lymph node-like structure functioned as a lymph node, ICG was administered to hLEC+mMSC tissue-transplanted LD mice (day 21). ICG fluorescence signals were detected in the lymph node-like structure in hLEC+mMSC tissue-transplanted LD mice (Fig. 5d), indicating ICG was delivered to the lymph node-like structure formed by the transplanted hLEC+mMSC tissue. Consistent with these results, lymph nodes or lymph node-like structures harvested from normal mice and hLEC+mMSC tissue- or hLEC+hMSC tissue-transplanted LD mice after ICG administration showed similar fluorescence intensities (Fig. 5e-g).

Therapeutic effect of CeLyT transplantation on lymphedema in LD mice

Because the CeLyTs restored lymphatic flow in LD mice, we treated lymphedema in LD mice by transplantation with various tissues. LD mice were prepared and various suspended cells and bioengineered tissues were used for transplantation (Supplementary Fig. 4a [top]). The percentage change in the thickness of paws and legs of LD mice to that of normal mice (no treatment) increased until days 5–7, reaching 20% and 6%, respectively, and then remained almost constant until day 21 (Fig. 6a-c). The suspended hLECs transplantation group and suspended hMSCs transplantation group had little therapeutic effect, which was equivalent to the LD mice group. However, the hLEC+mMSC and hLEC+hMSC tissue groups had a suppressive effect on the change ratio after a transient increase, which finally was almost equivalent to that of normal mice. Consistent with these results, sectioned images of paws showed that only hLEC+mMSC tissue and hLEC+hMSC tissue groups suppressed the accumulation of interstitial fluid in the paws of the LD mice (Fig. 6d). We then treated lymphedema after its formation in LD mice transplanted with hLEC+hMSC tissues on day 5 after LD (Supplementary Fig. 4a, bottom). Consistent with the above results, hLEC+hMSC tissues suppressed the increase in the percentage change in the thickness of paws and legs in LD mice, inhibited the accumulation of interstitial fluid, and promoted ICG influx into the lymph node-like structure formed at the transplantation site (Supplementary Fig. 4b-e). To confirm the therapeutic effect of CeLyTs on lymphedema, mMSC tissues and hLEC/mMSC mixed tissues prepared by single seeding without centrifugation were transplanted into LD mice. Although these tissues formed lymph node-like structures, the ICG influx into the lymph node-like structure, the therapeutic effect on lymphedema, and the suppressive effect on the accumulation of interstitial fluid were poor (Supplementary Fig. 5a-e). These findings suggested CeLyTs containing LECs formed functional lymph node-like structures and had a marked therapeutic effect on lymphedema.

Analyses of lymph node-like structures after CeLyT transplantation

Finally, we analyzed the characteristics of lymph node-like structures formed after the transplantation of CeLyTs. We evaluated whether cells forming lymph node-like structures were derived from the transplanted CeLyTs, the host animals, or both. Immunostaining of CD31 and Prox-1 using different cross-species antibodies (human and mouse) showed that the lymph node-like structures formed by hLEC+hMSC tissue transplantation were composed of cells derived from the hLEC+hMSC tissues and host animal, indicating CeLyTs induced the formation of lymph node-like structures in mice after transplantation (Fig. 7a). If these lymph node-like structures function as lymph nodes, immune cells must be present. Therefore, we immunostained lymph node-like structures formed after hLEC+hMSC tissue transplantation, with CD3 (T cells), B220 (B cells), CD11b (macrophages), and CD11c (dendritic cells) antibodies, and confirmed their presence (Fig. 7b and Supplementary Fig. 6a-c). Interestingly, B cells within the lymph node-like structures formed a follicle-like structure, similar to that in the lymph nodes of normal mice (Supplementary Fig. 6a). In addition, we evaluated the immune responses of the lymph node-like structures using immunostimulatory CpG1018. The lymph node-like structures produced various proinflammatory cytokines including interferon (IFN)-g, interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-a, the levels of which were equivalent to those in the lymph nodes of normal mice (Fig. 7c). To confirm the function of the lymph node-like structures as lymph nodes, ICG influx was also analyzed after ICG administration to hLEC+hMSC tissue-transplanted mice (day 21) by in vivo imaging. ICG fluorescence signals at the transplantation site of hLEC+hMSC tissues increased with time starting 10 min after ICG administration, and then the signals migrated to the adjacent lymph nodes, indicating ICG that flowed into the lymph node-like structure was subsequently drained (Supplementary Fig. 6d). In addition, the lymph node-like structure remained 100 days after the transplantation of CeLyTs (data not shown).

This study successfully fabricated CeLyTs composed of LECs and MSCs and demonstrated its usefulness for the treatment of secondary lymphedema, for which no effective radical treatment is available. Many previous reports have attempted to develop cell-based therapy for lymphedema and have reported excellent results, such as the formation of the lymphatic vessels in vivo [18, 19, 27, 28]. However, the reconstruction or regeneration of lymph nodes or functional lymph node-like structures and their therapeutic effects on lymphedema have never been achieved. To achieve this, we focused on the survival and presence of functional LECs in a three-dimensional cellular structure after transplantation. The centrifugal cell stacking technique can fabricate a three-layered cellular structure composed of an internal layer of LECs and two external layers of MSCs, which can prolong the survival of LECs after transplantation without unnecessary additions and severe cell damage. In addition, this structure can induce the formation of a lymphatic network of LECs in the structure. Then, CeLyTs form a lymph node-like structure after transplantation into mice, resulting in the restoration of lymph node functions in LD mice.

For cell-based therapy, cell survival is an important factor related to the therapeutic effect, as we previously reported [29–31]. This is because the transplanted cells release therapeutic cytokines and contribute to the reconstruction of the tissues themselves. In most cases, immunosuppressive drugs such as tacrolimus are used to prolong the survival of transplanted cells in cell-based therapy [32–34]. However, the long-term use of immunosuppressive drugs causes systemic adverse effects such as renal disorder, myelosuppression, and increased risk of infectious diseases, leading to a reduced quality of life for patients [35–37]. MSCs are multipotent stromal cells that can differentiate into multiple lineages and support cell survival by releasing immunosuppressive cytokines without systemic side effects in the case of local transplantation. We hypothesized that a three-dimensional cellular structure composed of MSCs on the outside would have long-term survival. Indeed, previous reports showed the long-term survival of a sheet-like cellular structure composed of MSCs [38–40]. Here, mMSC/Nluc + hMSC, hLEC + mMSC/Nluc, and mMSC tissues had long-term survival and formed lymph node-like structures after transplantation, whereas suspended mMSCs or mMSC/Nluc + NIH3T3 tissues disappeared within a few days (Fig. 4a-c, Fig. 5b,c). Furthermore, we speculated that the survival of the internal cells of CeLyTs was more important for the treatment of lymphedema, because the mMSC tissues or hLEC/mMSC mixed tissues prepared by single seeding without centrifugation had a poor therapeutic effect on lymphedema (Supplementary Fig. 5c-e). This result indicated that LECs were necessary to reconstruct the lymph node-like structures that functioned as lymph nodes. We evaluated the survival of internal cells in the respective bioengineered tissues, and demonstrated that the survival of the internal cells in CeLyTs (mMSC/Nluc + mMSC tissues or hLEC/CFSE + hMSC tissues) was maintained longer compared with that in the hLEC/CFSE/hMSC mixed tissues (Fig. 3c and Supplementary Fig. 2c). Therefore, the presence of MSCs in the bioengineered lymphatic tissues and the centrifugal cell stacking technique contributed to the long-term survival of LECs after the transplantation of CeLyTs.

Previously reported three-dimensional cellular structures containing LECs promoted an internal lymphatic network in the structure [21, 41, 42]. In general, endothelial cells, including lymphatic endothelial cells, can form a vascular network related to external topological effects and the presence of surrounding collagen, which exerts the original functions [26, 43, 44]. Therefore, we cultured the fabricated bioengineered tissues for 5 days to form a lymphatic network in the tissue (Fig. 3b and Supplementary Fig. 1d, e). The immunostaining of collagen 1A2 in the bioengineered tissue showed that mMSCs played the role of collagen 1A2 donor and mMSC sheets with high expression of collagen 1A2 provided the collagen 1A2-rich environment (Supplementary Fig. 1d, e). In addition, the expressions of lymphatic endothelial cell marker genes, PDPN and VEGFR-3, in the hLEC + mMSC tissues increased with time during culture (Supplementary Fig. 2b), and were higher than those in the hLEC/mMSC mixed tissue (Supplementary Fig. 2a). These results indicated that the centrifugal cell stacking technique induced the formation of a lymphatic network in the structure, and the appropriate culture time led to the functionalization of LECs, resulting in the formation of functional lymph node-like structures after the transplantation of CeLyTs.

Lymph nodes are characterized as an active immune response by abundant immune cells and lymphatic inflow and outflow [45–48]. The transplantation of CeLyTs into LD mice formed a lymph node-like structure containing a wide range of immune cells, which promoted an immune response to CpG1018, and the inflow and outflow of ICG (Fig. 7b, c and Supplementary Fig. 6a-d). These results indicate that the lymph node-like structure functions as a lymph node. Because the structure was composed of transplant- and host animal-derived cells (Fig. 7a), the bioengineered tissues can induce the formation of functional lymph node-like structures, and LECs in the tissues are the key to lymph node regeneration. In addition, lymph nodes do not regenerate in LD mice and suspended LECs barely formed lymph node-like structures (Supplementary Fig. 3b), indicating that the long-term survival of LECs or presence of MSCs with LECs may be essential for the formation of functional lymph node-like structures. Of note, CeLyTs did not show an immediate therapeutic effect in LD mice, which occurred 7–10 days after transplantation. Thus, CeLyTs might initially induce lymphangiogenesis with angiogenesis around the transplantation site, leading to the establishment of an immature lymph node-like structure formed by incorporating host animal-derived cells into the tissue within several days, followed by its maturation and ability to function as a lymph node 7–10 days after transplantation. During this process, various immune cells migrate to the lymph node-like structure through the newly formed lymphatic vessels that form a vascular network in the structure. A more detailed study of this hypothesis is required in the future.

To achieve the radical treatment of lymphedema, the reconstruction or regeneration of functional lymph nodes is required. CeLyTs composed of LECs and MSCs might reconstruct lymph nodes in LD patients and thus be a promising therapy for secondary lymphedema.

Materials and animals

The mouse mesenchymal stem cell line C3H10T1/2 (mMSC) was kindly provided by Dr. Hiroki Kagawa (Kyoto, Japan). The mouse fibroblast cell line NIH3T3 (fibroblast) was obtained from RIKEN Bioresource Center (RIKEN BRC) (Ibaraki, Japan). Human primary adipose-derived stem cells (hMSC) were kindly provided by Rohto Pharmaceutical Co., Ltd. (Osaka, Japan). Human primary lymphatic endothelial cells (hLEC) were purchased from Takara Bio Inc. (Tokyo, Japan). All other chemicals used were of the highest commercially available grade. Male nude BALB/c Slc-nu/nu mice (5–8-week old) were purchased from Sankyo Labo Service Co., Inc. (Tokyo, Japan) and maintained under specific pathogen-free conditions. All animals were treated under anesthesia using isoflurane, and euthanized by excess anesthesia after experiments. The protocols for animal experimentations were conducted in accordance with the Institutional Animal Experimentation Committee of the Tokyo University of Science (latest approval number: Y23004, latest approval date: August 8, 2024). All animal experimentations were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines.

Cell culture

mMSCs, firefly luciferase (fluc) gene-expressing mMSCs (mMSC/fluc), NanoLuc luciferase (Nluc) gene-expressing mMSCs (mMSC/Nluc), and green fluorescent protein (GFP) gene-expressing mMSCs (mMSC/GFP) cells were cultured in DMEM supplemented with 15% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine solution. NIH3T3 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin-L-glutamine solution. hMSCs were cultured in R-STEM Medium for hMSC High Growth (Rohto Pharmaceutical Co., Ltd.). hLECs were cultured in Endothelial Cell Media MV2 (Takara Bio Inc.) and maintained in a humidified atmosphere containing 5% CO₂ at 37°C.

Fabrication of bioengineered lymphatic tissues by cell stacking

mMSCs, hMSCs, mMSC/GFP, mMSC/Nluc, carboxyfluorescein succinimidyl ester (CFSE)-labeled mMSCs (mMSC/CFSE), or NIH3T3 (7.5×10⁵ cells) were collected with a trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Nacalai Tesque Inc., Kyoto, Japan), seeded into 24-well Transwell inserts (Corning, NY, USA) and set into a culture plate. The culture plate with seeded cells was immediately centrifuged under various conditions (centrifugation speed from 50 ×g to 1000 ×g and centrifugation time from 10 sec to 3 min) or 750 ×g and 30 sec to settle the cells at the bottom of the inserts. After 24 h-incubation, hLECs, hLEC/CFSE, mMSC/Nluc, and mMSC/fluc (2×10⁵ cells) were gently seeded on the layered MSCs and settled down on the layered cells in the inserts using the same procedure. After another 24 h-incubation, mMSCs, hMSCs, mMSC/GFP, and mMSC/Nluc or mMSC/CFSE (7.5×10⁵ cells) were gently seeded on LECs and MSCs and settled down on the layered cells in the inserts using the same procedure. Then, the cells were cultured for 5 days with changes of medium.

Fabrication of bioengineered tissues by cell coating

Fibronectin and gelatin coating of cells was performed as previously reported [20]. Briefly, NIH3T3 cells (1×10⁶ cells) or mMSCs/Nluc (2×10⁵ cells) were collected with a trypsin-EDTA solution and suspended in 0.04 mg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM Tris-HCl buffer (pH 7.4) and incubated for 1 min with gentle rotation. Subsequently, the cells were suspended in 0.04 mg/mL of gelatin (Sigma-Aldrich) in 50-mM Tris-HCl buffer at pH 7.4, and incubated for 1 min with gentle rotation. After each procedure, the cells were washed with 50 mM Tris-HCl buffer (pH 7.4). This procedure was repeated four times, and cells were finally coated with fibronectin. After suspending the fibronectin and gelatin-coated NIH3T3 cells in DMEM, they were seeded into Transwell inserts. After 24 h-incubation, the fibronectin and gelatin-coated mMSCs/Nluc were seeded on the fibronectin and gelatin-coated NIH3T3 cells and attached to the bottom of the inserts by the same procedure. After another 24 h-incubation, the fibronectin and gelatin-coated NIH3T3 cells were gently seeded on the fibronectin and gelatin-coated mMSCs/Nluc and attached to the bottom of the inserts using the same procedure. Then, the cells were cultured for 5 days with changes of medium.

Characterization of the bioengineered tissues

The survival of cells in the bioengineered tissues was evaluated by analyzing CFSE stained cells, measuring the luciferase activity of Nluc in the culture supernatant, and in vivo imaging of fluc-expressing cells. For CFSE staining, cells were prepared by incubation in medium containing 10 µM CFSE for 30 min, and the bioengineered tissues containing CFSE stained cells were observed with a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan). To measure luciferase activity, the culture supernatants of bioengineered tissues containing Nluc-expressing cells were collected daily and their luciferase activity was measured with a 2104 EnVision multilabel plate reader (PerkinElmer, Waltham, MA, USA). For in vivo imaging, the luminescence signals of bioengineered tissues containing fluc-expressing cells were detected with VivoGLo Luciferin (Promega, Madison, WI, USA) using a bioluminescence imaging system (In-Vivo Xtreme II, Bruker, Billerica, MA, USA). The structure of the engineered tissues was evaluated by observing the bioengineered tissue composed of CellTracker™ Green (ThermoFisher Scientific, Inc., Waltham, MA, USA)-labeled mMSCs and CellTracker™ Orange (ThermoFisher Scientific, Inc.)-labeled hLECs using a Leica SP8 laser scanning confocal microscope with LAS X Life Science software (Leica Microsystems, Wetzlar, Germany) or a BZ-X800 fluorescence microscope (Keyence). mMSCs and hLECs were prepared by incubation with medium containing 10 µM CellTracker™ Green or CellTracker™ Orange for 30 min, respectively.

Preparation of paraffin sections and cryosections

Bioengineered tissues fabricated in Transwell inserts were fixed with 4% paraformaldehyde (PFA). The bottoms of the Transwell inserts were cut out, embedded in paraffin, and sectioned at 5 µm thickness. The preparation of paraffin sections was performed by Biopathology Institute Co. Ltd. (Oita, Japan). The paraffin sections were observed using a BZ-X800 fluorescence microscope (Keyence). Mouse skin containing the transplanted bioengineered tissues was excised using scissors, embedded in O.C.T. compound (Sakura Finetek, Torrance, CA, USA), and frozen using liquid nitrogen. The frozen tissues were stored at − 80°C until use. The frozen sections were cut to 7 µm thickness using a CM3050 S cryostat (Leica Biosystems, Wetzler, Germany).

Immunohistochemical staining

Bioengineered tissues and cryo-sectioned tissues were fixed with 4% PFA, permeabilized with methanol, dimethyl sulfoxide, and a hydrogen peroxide mixture (6:1:1), and blocked with 1% bovine serum albumin for immunofluorescence staining. Bioengineered tissues and cryo-sectioned tissues were stained using primary antibodies and corresponding secondary antibodies. Nuclei were stained with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Immunofluorescence images were captured using a Leica SP8 laser scanning confocal microscope with a LAS X Life Science software (Leica Microsystems). The primary and secondary antibodies were as follows: 1:200 PROX-1 (Proteintech, Rosemont, IL, USA, 11067-2-AP), 1:500 mouse PROX-1 (Abcam, Cambridge, UK, ab101851), 1:500 human PROX-1 (Bio-Techne, Minneapolis, MN, USA, AF2727), 1:500 CD31 (Bio-Techne, NB600-1475), 1:500 mouse CD31 (Abcam, ab256569), 1:500 human CD31 (Abcam, ab76533), 1:100 B220/CD45R (Bio-Techne, NBP2-53303), 1:100 CD3 (Arigo, Zhubei, Taiwan, ARG22819), 1:50 CD11b (Abcam ab8878), 1:50 CD11c (Thermo Fisher Scientific, PA5-90208), 1:500 Collagen Type I (Proteintech, 14695-1-AP), 1:500 anti-rabbit Alexa488 (Thermo Fisher Scientific, A21206), 1:500 anti-rat Alexa488 (Thermo Fisher Scientific, A-11006), 1:500 anti-rabbit Alexa647 (Thermo Fisher Scientific, A31573), and 1:500 anti-goat Alexa647 (Thermo Fisher Scientific, A21447).

Evaluation of bioengineered tissue survival after transplantation

Bioengineered tissues containing Nluc-expressing cells were subcutaneously transplanted to normal mice or the site of the removed popliteal lymph node in the right lower limb of LD mice. To detect the luminescence of cells, 5 mg Nano-Glo Luciferase Assay System (Promega) dissolved in 100 µL PBS was injected into the transplantation site of mice, and luminescence was detected using a bioluminescence imaging system (In-Vivo Xtreme II, Bruker). In addition, the intensity of the region of interest (ROI) signal at the transplantation site was quantified using Bruker Molecular Imaging software with an In-Vivo Xtreme II system (Bruker). For the quantitative evaluation of cell survival, blood was collected daily from the posterior facial vein of mice after transplantation of bioengineered tissues containing Nluc-expressing cells, and the luciferase activity in the blood was measured using a 2104 EnVision multilabel plate reader (PerkinElmer).

Evaluation of lymph flow and lymphedema size in LD mice

LD mice were prepared by removing the popliteal and inguinal lymph nodes in the right lower limbs of mice as previously described [49]. Briefly, skin near the inguinal region of the right lower limb of mice was cut and the popliteal and inguinal lymph nodes were removed. Bioengineered tissues, suspended mMSC/Nluc (2×10⁶ cells), or suspended hLEC (2×10⁶ cells) were transplanted to the site of the removed popliteal lymph node. Thirty µg indocyanine green (ICG, MP Biomedicals, Irvine, CA, USA) dissolved in 30 µL PBS was administered into the footpad of the right lower limb of mice 1, 14, and 21 days after transplantation of the bioengineered tissues. Then, fluorescence at the site of the removed popliteal lymph nodes (the transplantation site) was detected 1 h after ICG administration using In-Vivo Xtreme II (Bruker). In addition, lymph nodes or lymph node-like structures formed by transplantation of the bioengineered tissues were excised from normal mice or LD mice on day 21, and the fluorescence intensity of the homogenates was measured by a 2104 EnVision multilabel plate reader (PerkinElmer). For the qualitative evaluation of lymphedema of LD mice, the accumulation of interstitial fluid to the paws of LD mice with or without transplantation of cells or bioengineered tissues was observed by a BZ-X800 fluorescence microscope (Keyence). For quantitative evaluation, the thicknesses of the paws and legs of the right lower limbs of LD mice with or without transplantation of cells or bioengineered tissues were measured and the change rate to day 0 was calculated.

Measurement of cytokine production

Bioengineered tissues were transplanted to the site of the removed popliteal lymph nodes of right lower limbs in LD mice. After 21 days, 10 nmol CpG1018 (sequence: 5′-TGACTGTGAACGTTCGAGATGA-3′ with a phosphorothioate-backbone) was injected into the footpad. Three hours after injection, the popliteal lymph nodes or lymph node-like structures formed by transplantation of the bioengineered tissues with lymphatic network were excised and homogenized. Then, the amount of interferon (IFN)-γ, interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α in the homogenates was quantified using mouse IFN-γ, IL-6, and IL-12/IL-23 (p40), and TNF-α ELISA MAX Deluxe Sets (BioLegend, San Diego, CA, USA).

Statistical analysis

Statistical differences were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s test for multiple comparisons or Student’s t-test for comparisons between two groups. Statistical significance was set at p < 0.05.

Acknowledgements

We would like to thank Biopathology Institute Co., Ltd. for their kind support in some experiments, and Rohto Pharmaceutical Co., Ltd. for providing hMSCs. This work was supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (grant number 23H03749), a Project Seeds A from the Japan Agency for Medical Research and Development (AMED), and a Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan. We thank J. Ludovic Croxford, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Author contributions

M.M. and K.K. conceptualized this research. S.O. and K.K. performed the study investigation. S.O. performed the experiments. S.O., S.I., M.M., M.N., and K.K. reviewed the data. S.O., S.I., M.M., M.N., and K.K. wrote the manuscript. All authors read and revised the manuscript.

Competing interests

The authors declare no competing interests.

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Reconstruction of the lymphatic system by transplantation of a centrifuge-based bioengineered lymphatic tissue

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Abstract

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Introduction

Results

Discussion

Online Methods

Materials and animals

Cell culture

Fabrication of bioengineered lymphatic tissues by cell stacking

Fabrication of bioengineered tissues by cell coating

Characterization of the bioengineered tissues

Preparation of paraffin sections and cryosections

Immunohistochemical staining

Evaluation of bioengineered tissue survival after transplantation

Evaluation of lymph flow and lymphedema size in LD mice

Measurement of cytokine production

Statistical analysis

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References

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