OUT OF THE BAG ? FUNCTIONAL CHARACTERIZATION OF EX VIVO EXPANDED MSC
(Abstract release date: 05/19/16)
EHA Library. Lopes S. 06/09/16; 134743; PB1843
Mr. Sergio Lopes
Contributions
Contributions
Abstract
Abstract: PB1843
Type: Publication Only
Background
Bone marrow (BM) derived mononuclear cells (MNC) are the starting point of numerous protocols, such as ex vivo expansion of HPC, neuro and mesenchymal stem cells (MSC).We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Aims
In order to further validate the applicability of these cells, MSC obtained were tested for morphology, immunophenotype, ability to hamper alloreactivity and to differentiate into adipocytes. tyle='mso-ansi-language:EN-GB'>We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Methods
The collection bag and filter system were back-washed and rinsed with RPMI under aseptic conditions and MNC isolated by density gradient centrifugation.To obtain long term MSC cultures, MNC were cultured in DMEM media supplemented with foetal bovine serum, at 37ºC and 5% CO2. Cultures were replated when confluence was reached.MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Results
In all cases, filter recovered MNC viability was superior to 90%, and long term MSC cultures were established, as shown by morphology and immunophenotype (CD105+, CD44+, CD90+, CD73+, CD45-, CD14-, and CD34-).To further characterize our MSC lineage differentiation, a hallmark of MSC, was shown by Oil Red staining of MSC-derived adipocytes. Another characteristic of MSC, down regulation of alloreactivity, was demonstrated in vitro, with a consistent 20% reduction of proliferating cells. -family:Calibri;mso-bidi-font-family:'Times New Roman'; mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'>MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Conclusion
With this study we demonstrated that, from the usually discarted BM collection bag and filter system, long term MSC cultures can be established. These cells can be expanded ex vivo, maintaining an appropriate phenotype and functional capabilities, being suitable for both investigation and clinical settings.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell
Type: Publication Only
Background
Bone marrow (BM) derived mononuclear cells (MNC) are the starting point of numerous protocols, such as ex vivo expansion of HPC, neuro and mesenchymal stem cells (MSC).We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Aims
In order to further validate the applicability of these cells, MSC obtained were tested for morphology, immunophenotype, ability to hamper alloreactivity and to differentiate into adipocytes. tyle='mso-ansi-language:EN-GB'>We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Methods
The collection bag and filter system were back-washed and rinsed with RPMI under aseptic conditions and MNC isolated by density gradient centrifugation.To obtain long term MSC cultures, MNC were cultured in DMEM media supplemented with foetal bovine serum, at 37ºC and 5% CO2. Cultures were replated when confluence was reached.MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Results
In all cases, filter recovered MNC viability was superior to 90%, and long term MSC cultures were established, as shown by morphology and immunophenotype (CD105+, CD44+, CD90+, CD73+, CD45-, CD14-, and CD34-).To further characterize our MSC lineage differentiation, a hallmark of MSC, was shown by Oil Red staining of MSC-derived adipocytes. Another characteristic of MSC, down regulation of alloreactivity, was demonstrated in vitro, with a consistent 20% reduction of proliferating cells. -family:Calibri;mso-bidi-font-family:'Times New Roman'; mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'>MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Conclusion
With this study we demonstrated that, from the usually discarted BM collection bag and filter system, long term MSC cultures can be established. These cells can be expanded ex vivo, maintaining an appropriate phenotype and functional capabilities, being suitable for both investigation and clinical settings.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell
Abstract: PB1843
Type: Publication Only
Background
Bone marrow (BM) derived mononuclear cells (MNC) are the starting point of numerous protocols, such as ex vivo expansion of HPC, neuro and mesenchymal stem cells (MSC).We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Aims
In order to further validate the applicability of these cells, MSC obtained were tested for morphology, immunophenotype, ability to hamper alloreactivity and to differentiate into adipocytes. tyle='mso-ansi-language:EN-GB'>We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Methods
The collection bag and filter system were back-washed and rinsed with RPMI under aseptic conditions and MNC isolated by density gradient centrifugation.To obtain long term MSC cultures, MNC were cultured in DMEM media supplemented with foetal bovine serum, at 37ºC and 5% CO2. Cultures were replated when confluence was reached.MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Results
In all cases, filter recovered MNC viability was superior to 90%, and long term MSC cultures were established, as shown by morphology and immunophenotype (CD105+, CD44+, CD90+, CD73+, CD45-, CD14-, and CD34-).To further characterize our MSC lineage differentiation, a hallmark of MSC, was shown by Oil Red staining of MSC-derived adipocytes. Another characteristic of MSC, down regulation of alloreactivity, was demonstrated in vitro, with a consistent 20% reduction of proliferating cells. -family:Calibri;mso-bidi-font-family:'Times New Roman'; mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'>MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Conclusion
With this study we demonstrated that, from the usually discarted BM collection bag and filter system, long term MSC cultures can be established. These cells can be expanded ex vivo, maintaining an appropriate phenotype and functional capabilities, being suitable for both investigation and clinical settings.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell
Type: Publication Only
Background
Bone marrow (BM) derived mononuclear cells (MNC) are the starting point of numerous protocols, such as ex vivo expansion of HPC, neuro and mesenchymal stem cells (MSC).We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Aims
In order to further validate the applicability of these cells, MSC obtained were tested for morphology, immunophenotype, ability to hamper alloreactivity and to differentiate into adipocytes. tyle='mso-ansi-language:EN-GB'>We have previously established that the BM collection bag and filter system (which are usually discarded) are an alternative source of MNC, yielding viable and sterile cells equivalent to 50ml of filtered BM, and should not be considered clinical waste. The viability and functionality of the recovered MNC were evaluated by trypan blue exclusion and in vitro differentiation into MSC, respectively.
Methods
The collection bag and filter system were back-washed and rinsed with RPMI under aseptic conditions and MNC isolated by density gradient centrifugation.To obtain long term MSC cultures, MNC were cultured in DMEM media supplemented with foetal bovine serum, at 37ºC and 5% CO2. Cultures were replated when confluence was reached.MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Results
In all cases, filter recovered MNC viability was superior to 90%, and long term MSC cultures were established, as shown by morphology and immunophenotype (CD105+, CD44+, CD90+, CD73+, CD45-, CD14-, and CD34-).To further characterize our MSC lineage differentiation, a hallmark of MSC, was shown by Oil Red staining of MSC-derived adipocytes. Another characteristic of MSC, down regulation of alloreactivity, was demonstrated in vitro, with a consistent 20% reduction of proliferating cells. -family:Calibri;mso-bidi-font-family:'Times New Roman'; mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'>MSC morphology was confirmed with a reverse microscope, and immunophenotype by flow cytometry. To assess adipogenic differentiation capacity, MSC were cultured in the presence of dexamethasone and diclofenac, with adipocytes generation tested by Oil Red staining of lipid deposits. To evaluate their imunossupressive role a one way MLR was performed. Briefly, irradiated MNC (stimulator) and CFSE-dyed MNC (responder) were co-cultured in the presence or absence (control) of MSC. After 7 days of incubation, proliferation of responder cells was measured by flow cytometry.
Conclusion
With this study we demonstrated that, from the usually discarted BM collection bag and filter system, long term MSC cultures can be established. These cells can be expanded ex vivo, maintaining an appropriate phenotype and functional capabilities, being suitable for both investigation and clinical settings.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell
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