Perfusion-based Bioreactor Research Articles

Orthotopic transplantation of the bioengineered lung using a mouse-scale perfusion-based bioreactor and human primary endothelial cells

Whole lung engineering and the transplantation of its products is an ambitious goal and ultimately a viable solution for alleviating the donor-shortage crisis for lung transplants. There are several limitations currently impeding progress in the field with a major obstacle being efficient revascularization of decellularized scaffolds, which requires an extremely large number of cells when using larger pre-clinical animal models. Here, we developed a simple but effective experimental pulmonary bioengineering platform by utilizing the lung as a scaffold. Revascularization of pulmonary vasculature using human umbilical cord vein endothelial cells was feasible using a novel in-house developed perfusion-based bioreactor. The endothelial lumens formed in the peripheral alveolar area were confirmed using a transmission electron microscope. The quality of engineered lung vasculature was evaluated using box-counting analysis of histological images. The engineered mouse lungs were successfully transplanted into the orthotopic thoracic cavity. The engineered vasculature in the lung scaffold showed blood perfusion after transplantation without significant hemorrhage. The mouse-based lung bioengineering system can be utilized as an efficient ex-vivo screening platform for lung tissue engineering.

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.

Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements.

Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the "sandwich cultures", were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.

Abstract 6041: Perfusion-based bioreactor preserves microenvironment of hepatocellular carcinoma ex vivo

Abstract Background: Hepatocellular carcinoma (HCC) accounts for 75-85% of all primary liver malignancies. HCC represents an attractive target for immune checkpoint inhibitors (ICI) and tumor microenvironment (TME) agents. The development and success of ICI therapies requires a comprehensive model to study interactions of immune networks in the TME. Patient-derived organoids (PDO) have emerged as promising individualized models for ex vivo drug testing. While PDO are superior to previous models, due to their inherent heterogeneity and their enhanced capacity to capture tumor characteristics, the lack of mesenchymal and immune components limit their use for testing drug classes such as ICI or study tumor-TME interaction. Here, we developed and characterized a perfusion-based cultivated ex vivo HCC tissue model that preserves the tumor tissue architecture and maintains the pathophysiological microenvironment of human tissue for testing of TME-related treatments. Materials and methods: Fresh tumor tissue was obtained from surgical specimens of six patients undergoing liver resection for HCC. Tissue fragments of 2x2x2 mm were installed between two discs of collagen type 1 porous scaffold and placed in a perfusion-based bioreactor (U-shaped Culture Under Perfusion; U-CUP) for tissue engineering or cultured in static condition. At different time points, cultured tissue fragments were formalin-fixed and paraffin-embedded for downstream analysis. Cell viability and morphology was monitored on day 0, 5 and 7 by using hematoxylin and eosin staining. The changes in TME composition were monitored by immunohistochemistry using cell-type specific antibodies like CD34 for endothelial cells, CD3 for T-cells, alpha-smooth muscle actin (a-SMA) for fibroblasts and arginase-L for urea cycle. Results: Microscopic analysis revealed that, compare to static culture, the morphology and the viability of the cells were preserved during the culture period in the U-CUP. Tissue histology as well as the immunophenotypic pattern observed in the original tissue was maintained. Similarly, the presence of endothelial cells (CD34+) and fibroblasts (a-SMA+) were detected in the U-CUP cultured samples in a similar proportion to the original tumor samples. The presence of T-cells (CD3+) was stable at all time points. Compared to the samples cultured under static conditions, the U-CUP tended to show less necrosis during the seven-day course. Conclusion: Compared to PDO, U-CUP bioreactor tumor culture seems to preserve mesenchymal and immune components. This enables further investigation and assessment of TME-related treatments, such as ICI. Citation Format: Gabriel Fridolin Hess, Manuele Giuseppe Muraro, Mairene Coto-Llerena, Silvio Däster, Caner Ercan, Simone Muenst, Otto Kollmar, Salvatore Piscuoglio, Savas Deniz Soysal. Perfusion-based bioreactor preserves microenvironment of hepatocellular carcinoma ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6041.

Procurement and Decellularization of Rat Hindlimbs using an Ex Vivo Perfusion-based Bioreactor for Vascularized Composite Allotransplantation.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma.

Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma.

Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues.

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

Enhanced articular cartilage decellularization using a novel perfusion-based bioreactor method

Current decellularization methods for articular cartilages require many steps, various and high amounts of detergents, and a relatively long time to produce decellularized scaffolds. In addition, such methods often damage the essential components and the structure of the tissue. This study aims to introduce a novel perfusion-based bioreactor (PBB) method to decellularize bovine articular cartilages efficiently while reducing the harmful physical and chemical steps as well as the duration of the process. This leads to better preservation of the structure and the essential components of the native tissue. Firstly, a certain number of channels (Ø 180 μm) were introduced into both sides of cylindrical articular bovine cartilage disks (5 mm in diameter and 1 mm in thickness). Next, the disks were decellularized in the PBB and a shaker as the control. Using the PBB method resulted in ∼90% reduction of DNA content in the specimens, which was significantly higher than those of the shaker results with ∼60%. Also, ∼50% sulfated glycosaminoglycan (sGAG) content and ∼92% of the compression properties were maintained implying the efficient preservation of the structure and components of the scaffolds. Moreover, the current study indicated that the PBB specimens supported the adherence and proliferation of the new cells effectively. In conclusion, the results show that the use of PBB method increases the efficiency of producing decellularized cartilage scaffolds with a better maintenance of essential components and structure, while reducing the chemicals and steps required for the process. This will pave the way for producing close-to-natural scaffolds for cartilage tissue engineering.

Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity

Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.

Platelet-rich plasma and stromal vascular fraction cells for the engineering of axially vascularized osteogenic grafts.

Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. We have previously shown that axially vascularized osteogenic constructs, engineered by combining human stromal vascular fraction (SVF) cells and a ceramic scaffold, can revitalize necrotic bone of clinically relevant size in a rat model of AVN. For a clinical translation, the fetal bovine serum (FBS) used to generate such grafts should be substituted by a nonxenogeneic culture supplement. Human thrombin-activated platelet-rich plasma (tPRP) was evaluated in this context. SVF cells were cultured inside porous hydroxyapatite scaffolds with a perfusion-based bioreactor system for 5 days. The culture medium was supplemented with either 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bovine bone cylinders to mimic the treatment of a necrotic bone. A ligated vascular bundle was inserted into the constructs upon subcutaneous implantation in the groin of nude rats. After 1 and 8 weeks, constructs were harvested, and vascularization, host cell recruitment, and bone formation were analyzed. After 1 week in vivo, constructs were densely vascularized, with no difference between tPRP- and FBS-based ones. After 8 weeks, bone formation and vascularization was found in both tPRP- and FBS-precultured constructs. However, the amount of bone and the vessel density were respectively 2.2- and 1.8-fold higher in the tPRP group. Interestingly, the density of M2, proregenerative macrophages was also significantly higher (6.9-fold) following graft preparation with tPRP than with FBS. Our findings indicate that tPRP is a suitable substitute for FBS to generate vascularized, osteogenic grafts from SVF cells and could thus be implemented in protocols for clinical translation of this strategy towards the treatment of bone loss and AVN.

Perfusion-Based Bioreactor for the Decellularization and Recellularization of Ferret Tracheas

Pai, Albert C. MD; Lynch, Thomas J. PhD; Ievlev, Vitaly BS; Ahlers, Bethany BS; Engelhardt, John F. PhD; Rajnikant Parekh, Kalpaj MBBS, FACS Author Information

Abstract 4097: Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related death, often diagnosed at advanced stages. Conventional chemotherapeutic regimens have limited success rates, and a major challenge is represented by the lack of adequate in vitro models predicting patient responsiveness to defined treatments, possibly guiding therapeutic decisions. Non-malignant cells, including mesenchymal and immune cells, critically affect development, progression and drug responsiveness of human CRC. However, tumor drug responses are still evaluated on culture systems, such as 2D tumor cell monocultures or on tumor xenografts, which do not preserve all cellular components of in vivo tumor microenvironment.In this work, we have investigated the suitability of a perfusion-based bioreactor for 3D culture of primary CRC samples. Freshly excised CRC specimens were cut into fragments, inserted between two collagen type I sponges in a “sandwich-like” format and cultured for three days in a perfused-based system or under static conditions.We show that cultures under perfusion result in significantly higher maintenance of tissue integrity as compared to static cultures, with preservation of whole tumor microenvironment components, including cancer cells, mesenchymal stromal cells and a fraction of immune cells. Tumor tissues cultured under perfusion displayed an almost intact architecture with viable and proliferating tumor cells. Stromal cells were also maintained in proportions similar to those of original tumors and were fully viable, as indicated by their responsiveness to microenvironmental stimuli, such as IL-17. In addition, immune cells were also partially preserved, and were capable to release effector cytokines upon activation. Importantly, perfusion-based cultures proved suitable for testing sensitivity of primary tumor cells to chemotherapies currently in use for CRC and revealed heterogeneous responsiveness across different samples. [C.M. and M.G.M. contributed equally to this work.] Citation Format: Celeste Manfredonia, Manuele Giuseppe Muraro, Christian Hirt, Valentina Mele, Valeria Governa, Adam Papadimitropoulos, Silvio Daester, Savas D. Soysal, Raoul A. Droeser, Robert Mechera, Daniel Oertli, Raffaele Rosso, Martin Bolli, Luigi M. Terracciano, Giulio C. Spagnoli, Ivan Martin, Giandomenica Iezzi. Maintenance of primary human colorectal cancer microenvironment using a perfusion bioreactor-based 3D culture system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4097.

Abstract 10.55 PLATELET-RICH PLASMA VS. FETAL BOVINE SERUM IN ENGINEERING OF AXIALLY-VASCULARIZED OSTEOGENIC GRAFTS FROM HUMAN ADIPOSE-DERIVED CELLS TO TREAT AVASCULAR NECROSIS OF BONE

INTRODUCTION: Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. Engineered, axially vascularized, human adipose-derived stromal vascular fraction (SVF) cell based osteogenic constructs have been shown to revitalize necrotic bone of clinically-relevant size in a challenging rat model of AVN. To reduce regulatory difficulties towards clinical translation, fetal bovine serum (FBS) was substituted by thrombin-activated platelet-rich plasma (tPRP). METHODS: SVF cells from 5 lipoaspirate were isolated and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5 days. The medium was supplemented either with 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8 weeks, vascularization and bone formation were analyzed. RESULTS: After 1 week, neither maximal distance of vessels from the bundle nor vascular density was significantly different. However, FBS cultured grafts revealed significantly more human vessel (hCD34) and osteoclasts compared to tPRP cultured grafts. Overall macrophages and M2 macrophages were not different between FBS and tPRP cultured grafts. After 8 weeks in vivo, 3/5 samples from FBS culture and 1/5 sample from tPRP culture showed bone formation. Roughly 40% of the osteocytes were of human origin (36% from FBS and 42% from tPRP culture). DISCUSSION: Despite promising results in literature, replacement of FBS by tPRP resulted in a reduced capacity of bone formation in our series. Further studies are necessary to analyze and improve the low bone formation capacity, possibly caused by the heterogeneity of the SVF donors and tPRP samples.

Engineered, axially-vascularized osteogenic grafts from human adipose-derived cells to treat avascular necrosis of bone in a rat model.

Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. The standard of care, vascularized bone grafts, is limited by donor site morbidity and restricted availability. The aim of this study was to generate and test engineered, axially vascularized SVF cells-based bone substitutes in a rat model of AVN. SVF cells were isolated from lipoaspirates and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5days. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8weeks in vivo, bone formation and vascularization were analyzed. Newly-formed bone was found in 80% of SVF-seeded scaffolds after 8weeks but not in unseeded controls. Human ALU+cells in the bone structures evidenced a direct contribution of SVF cells to bone formation. A higher density of regenerative, M2 macrophages was observed in SVF-seeded constructs. In both experimental groups, devitalized bone was revitalized by vascularized tissue after 8 weeks. SVF cells-based osteogenic constructs revitalized fully necrotic bone in a challenging AVN rat model of clinically-relevant size. SVF cells contributed to accelerated initial vascularization, to bone formation and to recruitment of pro-regenerative endogenous cells. Avascular necrosis (AVN) of bone often requires surgical treatment with autologous bone grafts, which is surgically demanding and restricted by significant donor site morbidity and limited availability. This paper describes a de novo engineered axially-vascularized bone graft substitute and tests the potential to revitalize dead bone and provide efficient new bone formation in a rat model. The engineering of an osteogenic/vasculogenic construct of clinically-relevant size with stromal vascular fraction of human adipose, combined to an arteriovenous bundle is described. This construct revitalized and generated new bone tissue. This successful approach proposes a novel paradigm in the treatment of AVN, in which an engineered, vascularized osteogenic graft would be used as a germ to revitalize large volumes of necrotic bone.

Abstract 4833: Perfusion-based bioreactor culture of primary cancer tissue maintains tumor microenvironment complexity and allow in-vitro testing of immune blockade therapy

Abstract In vitro culture of primary cancer tissue is still very limited and the generation of patient derived xenograft determine the loss of human-cancer associated stroma. In this context, the use of 3D in vitro systems based on human tissue may be an innovative system to be exploited for keeping the tumor microenvironment (TME) complexity of the tissue in vitro. Freshly excised colorectal (CRC) and breast cancer (BrCa) specimens were fragmented and cultured in 3D “sandwich-like format” between porous collagen scaffolds under perfusion flow (U-CUP, Cellec Biotek AG). The maintenance of tumor and immune-infiltrating cells, survival and phenotypic characterization were histologically assessed. In a second step cancer treatment were tested. U-CUP culture allowed the preservation, viability and expansion of tumor tissue with concomitant stromal and immune cells. Expanding cancer cells were viable after 10 and 21 days (CRC and BrCa, respectively). Administration of anti-ER treatment to Lumina A ER+ BrCa was associated with decreased expansion of cancer tissue into the scaffold after 21 days. The maintenance of immune-infiltrating cells allowed testing of immune blockade therapy. Administration of anti-PDL1 antibody, alone or in combination with anti-CTLA4, to the culture medium was associated with increased expression of markers of immune-activation (i.e. IFNγ) and decreased expression of immunosuppressive cytokine IL10. Preserving malignant, interstitial and immunocompetent cells comprised in surgically excised tumor specimens might allow a direct evaluation of the effects of various treatments on the complex TME. This engineered in vitro model could allow animal-free testing and it could be extended as a platform allowing the testing of innovative approaches for the treatment of human malignancies. Our findings shed the light on a promising system for selecting personalized treatment based on a patient’s tumor specific microenvironment. Note: This abstract was not presented at the meeting. Citation Format: Manuele Giuseppe Muraro, Simone Muenst, Celeste Manfredonia, Valentina Mele, Silvio Daester, Alexandar Tzankov, Luigi Terracciano, Walter Weber, Giulio C. Spagnoli, Giandomenica Iezzi, Ivan Martin, Savas D. Soysal. Perfusion-based bioreactor culture of primary cancer tissue maintains tumor microenvironment complexity and allow in-vitro testing of immune blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2017-4833

Ex-vivo assessment of drug response on breast cancer primary tissue with preserved microenvironments

ABSTRACTInteraction between cancerous, non-transformed cells, and non-cellular components within the tumor microenvironment plays a key role in response to treatment. However, short-term culture or xenotransplantation of cancer specimens in immunodeficient animals results in dramatic modifications of the tumor microenvironment, thus preventing reliable assessment of compounds or biologicals of potential therapeutic relevance.We used a perfusion-based bioreactor developed for tissue engineering purposes to successfully maintain the tumor microenvironment of freshly excised breast cancer tissue obtained from 27 breast cancer patients and used this platform to test the therapeutic effect of antiestrogens as well as checkpoint-inhibitors on the cancer cells.Viability and functions of tumor and immune cells could be maintained for over 2 weeks in perfused bioreactors. Next generation sequencing authenticated cultured tissue specimens as closely matching the original clinical samples. Anti-estrogen treatment of cultured estrogen receptor positive breast cancer tissue as well as administration of pertuzumab to a Her2 positive breast cancer both had an anti-proliferative effect. Treatment with anti-programmed-death-Ligand (PD-L)-1 and anti-cytotoxic T lymphocyte-associated protein (CTLA)-4 antibodies lead to immune activation, evidenced by increased lymphocyte proliferation, increased expression of IFNγ, and decreased expression of IL10, accompanied by a massive cancer cell death in ex vivo triple negative breast cancer specimens.In the era of personalized medicine, the ex vivo culture of breast cancer tissue represents a promising approach for the pre-clinical evaluation of conventional and immune-mediated treatments and provides a platform for testing of innovative treatments.

Recellularization on Acellular Lung Tissue Scaffold Using Perfusion-Based Bioreactor: An Online Monitoring Strategy

In this study, we improved a perfusion-based bioreactor (PBB) system to recellularize acellular lung scaffolds. A method was used for the online monitoring of the recellularization process in order to find more data during a re-seeding procedure. Lungs were harvested from young adult (2-month-old) male rats. After preparation and pre-decellularization, the lungs were decellularized with liquid detergent in order to obtain an acellular scaffold. Acelluar tissues were stained by hematoxylin and eosin to verify efficient removal of cell components and debris. Furthermore, the maintenance of the extracellular matrix architecture was evaluated using scanning electron microscopy. After rinsing for detergent removal, human umbilical cord vein endothelial cells were cultured on acellular tissue using a PBB. Data analysis of the dissolved oxygen through culturing indicated a specific growth rate of 0.0199 h−1 (doubling time, td = 34 h). The average oxygen uptake rate per cell was measured as 14.3 nmol/min per 106 cells. This study demonstrates that recellularization on acellular tissue can be monitored online to provide insight into the growth behavior of cells. The results show the high performance design of PBBs for tissue-engineered lungs.

Abstract 615: Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine

Abstract INTRODUCTION Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell responses to drugs but they are not suited for the development of immune-mediate therapies. Here we present an innovative method for in vitro culture primary breast cancer (BrCa) tissues in porous 3D scaffolds by using a perfusion-based bioreactor system (U-CUP). MATERIALS &amp; METHODS Freshly excised breast cancer specimens were fragmented and cultured in a 3D “sandwich-like format” between collagen porous scaffolds under perfusion flow. DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. Malignant and non-malignant cells survival, expansion into the scaffold and the ability to recapitulate features of the original BrCa specimens were histologically assessed. For estrogen receptor (ER) positive tissues we tested the response to hormonal therapy by adding the anti-ER drug Fulvestrant. Furthermore, the maintenance of immune-infiltrating cells allowed testing immune blockade therapy in vitro using anti PD-L1 on PD-L1 positive samples. Response to treatment was evaluated by histology and qRT-PCR for markers of immune-response. RESULTS By culturing BrCa using the U-CUP we were able to preserve viability and to promote the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Expanding cancer cells were viable after 21 days and recapitulating the initial histology with formation of glands. Administration of anti-ER treatment was associated with decreased expansion of cancer tissue into the scaffold after 21 days. After 7 days of anti PD-L1 antibody treatment we observed a reduced number of tumor cells due to the activation of infiltrating lymphocytes, as shown by increased expression of IFNg and decreased expression of IL10. CONCLUSIONS The scaffold-based perfusion bioreactor represents a successful organotypic tumor model allowing in vitro long-term culture of breast cancer specimens. Our findings shed the light on a promising system for selecting personalized treatment based on a patient's tumor specific microenvironment. Citation Format: Manuele Giuseppe Muraro, Simone Muenst, Valentina Mele, Luca Quagliata, Alexandar Tzankov, Walter P. Weber, Giulio C. Spagnoli, Savas D. Soysal. Scaffold-based perfusion bioreactor system for in vitro maintenance of primary breast cancer tissue microenvironment suitable for personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 615.

Abstract P4-04-19: Primary breast cancer culture in a perfusion-based bioreactor suitable for in vitro testing of immune blockade therapy

Abstract Introduction: Interaction between cancer cells and immune system critically affects development, progression and treatment of human malignancies. Two-dimensional (2D) in vitro culture systems and in vivo animal models are the primary tools used to test cancer cell response to drugs but they are not suited for the development of immune-mediated therapies. Here we present an innovative method to culture breast cancer tissue in porous 3D scaffolds by using a perfusion-based bioreactor system that allows the maintenance and expansion of tumor microenvironment. Experimental procedures: Freshly excised breast cancer specimens were fragmented and cultured in a 3D "sandwich-like format" between two layers of porous collagen scaffold under perfusion flow (U-CUP). DMEM/F12, supplemented with 10% autologous human serum, was used as a culture medium. We assessed the ability of tumor and non-malignant cells to survive and expand into the scaffold in perfusion culture, as well as their capacity to recapitulate features of the original breast cancer tissue. The maintenance of immune-infiltrating cells allowed testing of immune blockade therapy in vitro using anti-PD-L1 and anti-CTLA4 antibodies alone or in combination. Results: The U-CUP culture system preserved tissue viability better compared to a static culture and promoted the expansion of breast cancer cells from surgical specimens together with accompanying stromal and immune cells into the porous scaffold. Tumor tissues were viable after 21 days and mostly recapitulating the initial histology with formation of glands. Administration of anti-PDL1 antibody, alone or in combination with anti-CTLA4, to the culture medium was associated with increased expression of markers of immune-activation (i.e. IFNg) and decreased expression of immunosuppressive cytokine IL10. Conclusions: Our results show that culture of breast cancer tissue in a 3D perfusion-based bioreactor might represent a promising system for the pre-clinical evaluation of immune-mediated therapies. Preserving malignant, interstitial and immunocompetent cells comprised in surgically excised breast cancer samples might allow a direct evaluation of the effects of various treatments on the complex tumor microenvironment. This engineered in vitro model could be extended as a platform allowing the testing of innovative approaches for the treatment of human malignancies, possibly in the direction of personalized medicine. Citation Format: Muraro MG, Muenst S, Mele V, Spagnoli GC, Oertli D, Weber WP, Soysal SD. Primary breast cancer culture in a perfusion-based bioreactor suitable for in vitro testing of immune blockade therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-19.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment.