10 research outputs found
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 40â60%. 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 21Â 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)
Therapeutic microparticles functionalized with biomimetic cardiac stem cell membranes and secretome
Stem cell therapy represents a promising strategy in regenerative medicine. However, cells need to be carefully preserved and processed before usage. In addition, cell transplantation carries immunogenicity and/or tumourigenicity risks. Mounting lines of evidence indicate that stem cells exert their beneficial effects mainly through secretion (of regenerative factors) and membrane-based cellâcell interaction with the injured cells. Here, we fabricate a synthetic cell-mimicking microparticle (CMMP) that recapitulates stem cell functions in tissue repair. CMMPs carry similar secreted proteins and membranes as genuine cardiac stem cells do. In a mouse model of myocardial infarction, injection of CMMPs leads to the preservation of viable myocardium and augmentation of cardiac functions similar to cardiac stem cell therapy. CMMPs (derived from human cells) do not stimulate T-cell infiltration in immuno-competent mice. In conclusion, CMMPs act as âsynthetic stem cellsâ which mimic the paracrine and biointerfacing activities of natural stem cells in therapeutic cardiac regeneration
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
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
Derivation of therapeutic lung spheroid cells from minimally invasive transbronchial pulmonary biopsies
Abstract 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 âlung spheroid cellsâ (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)
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
polyÂ(<i>N</i>-isopropylacrylamine-<i>co</i>-acrylic
acid) or PÂ(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