Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40–60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals

Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies

BACKGROUND: Resident stem and progenitor cells have been identified in the lung over the last decade, but isolation and culture of these cells remains a challenge. Thus, although these lung stem and progenitor cells provide an ideal source for stem-cell based therapy, mesenchymal stem cells (MSCs) remain the most popular cell therapy product for the treatment of lung diseases. Surgical lung biopsies can be the tissue source but such procedures carry a high risk of mortality. METHODS: In this study we demonstrate that therapeutic lung cells, termed "lung spheroid cells" (LSCs) can be generated from minimally invasive transbronchial lung biopsies using a three-dimensional culture technique. The cells were then characterized by flow cytometry and immunohistochemistry. Angiogenic potential was tested by in-vitro HUVEC tube formation assay. In-vivo bio- distribution of LSCs was examined in athymic nude mice after intravenous delivery. RESULTS: From one lung biopsy, we are able to derive >50 million LSC cells at Passage 2. These cells were characterized by flow cytometry and immunohistochemistry and were shown to represent a mixture of lung stem cells and supporting cells. When introduced systemically into nude mice, LSCs were retained primarily in the lungs for up to 21 days. CONCLUSION: Here, for the first time, we demonstrated that direct culture and expansion of human lung progenitor cells from pulmonary tissues, acquired through a minimally invasive biopsy, is possible and straightforward with a three-dimensional culture technique. These cells could be utilized in long-term expansion of lung progenitor cells and as part of the development of cell-based therapies for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF)

Intravenous Cardiac Stem Cell-Derived Exosomes Ameliorate Cardiac Dysfunction in Doxorubicin Induced Dilated Cardiomyopathy

Despite the efficacy of cardiac stem cells (CSCs) for treatment of cardiomyopathies, there are many limitations to stem cell therapies. CSC-derived exosomes (CSC-XOs) have been shown to be responsible for a large portion of the regenerative effects of CSCs. Using a mouse model of doxorubicin induced dilated cardiomyopathy, we study the effects of systemic delivery of human CSC-XOs in mice. Mice receiving CSC-XOs showed improved heart function via echocardiography, as well as decreased apoptosis and fibrosis. In spite of using immunocompetent mice and human CSC-XOs, mice showed no adverse immune reaction. The use of CSC-XOs holds promise for overcoming the limitations of stem cells and improving cardiac therapies

Intravenous Cardiac Stem Cell-Derived Exosomes Ameliorate Cardiac Dysfunction in Doxorubicin Induced Dilated Cardiomyopathy

Despite the efficacy of cardiac stem cells (CSCs) for treatment of cardiomyopathies, there are many limitations to stem cell therapies. CSC-derived exosomes (CSC-XOs) have been shown to be responsible for a large portion of the regenerative effects of CSCs. Using a mouse model of doxorubicin induced dilated cardiomyopathy, we study the effects of systemic delivery of human CSC-XOs in mice. Mice receiving CSC-XOs showed improved heart function via echocardiography, as well as decreased apoptosis and fibrosis. In spite of using immunocompetent mice and human CSC-XOs, mice showed no adverse immune reaction. The use of CSC-XOs holds promise for overcoming the limitations of stem cells and improving cardiac therapies

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals

Heart Repair Using Nanogel-Encapsulated Human Cardiac Stem Cells in Mice and Pigs with Myocardial Infarction

Stem cell transplantation is currently implemented clinically but is limited by low retention and engraftment of transplanted cells and the adverse effects of inflammation and immunoreaction when allogeneic or xenogeneic cells are used. Here, we demonstrate the safety and efficacy of encapsulating human cardiac stem cells (hCSCs) in thermosensitive poly­(<i>N</i>-isopropylacrylamine-<i>co</i>-acrylic acid) or P­(NIPAM-AA) nanogel in mouse and pig models of myocardial infarction (MI). Unlike xenogeneic hCSCs injected in saline, injection of nanogel-encapsulated hCSCs does not elicit systemic inflammation or local T cell infiltrations in immunocompetent mice. In mice and pigs with acute MI, injection of encapsulated hCSCs preserves cardiac function and reduces scar sizes, whereas injection of hCSCs in saline has an adverse effect on heart healing. In conclusion, thermosensitive nanogels can be used as a stem cell carrier: the porous and convoluted inner structure allows nutrient, oxygen, and secretion diffusion but can prevent the stem cells from being attacked by immune cells

Mesenchymal Stem Cell/Red Blood Cell-Inspired Nanoparticle Therapy in Mice with Carbon Tetrachloride-Induced Acute Liver Failure

Acute liver failure is a critical condition characterized by global hepatocyte death and often time needs a liver transplantation. Such treatment is largely limited by donor organ shortage. Stem cell therapy offers a promising option to patients with acute liver failure. Yet, therapeutic efficacy and feasibility are hindered by delivery route and storage instability of live cell products. We fabricated a nanoparticle that carries the beneficial regenerative factors from mesenchymal stem cells and further coated it with the membranes of red blood cells to increase blood stability. Unlike uncoated nanoparticles, these particles promote liver cell proliferation <i>in vitro</i> and have lower internalization by macrophage cells. After intravenous delivery, these artificial stem cell analogs are able to remain in the liver and mitigate carbon tetrachloride-induced liver failure in a mouse model, as gauged by histology and liver function test. Our technology provides an innovative and off-the-shelf strategy to treat liver failure