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Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

Laner-Plamberger S, Lener T, Schmid D, Streif DA, Salzer T, Öller M, Hauser-Kronberger C, Fischer T, Jacobs VR, Schallmoser K, Gimona M, Rohde E - J Transl Med (2015)

Bottom Line: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained.Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium.Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. s.laner-plamberger@salk.at.

ABSTRACT

Background: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin.

Methods: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages.

Results: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure.

Conclusions: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

No MeSH data available.


Related in: MedlinePlus

Experimental workflow: Isolation and characterization of umbilical cord (UC-) and bone marrow (BM-) MSCs. UC-MSCs group 1 (left) and BM-MSCs, group3 (right) were isolated using medium condition A exclusively. UC-MSCs group 2 (middle) were isolated in all three medium conditions (A, white; B, grey and C, black). After isolation and expansion, all MSC types tested by flow cytometric analysis and differentiation assays. Proliferation and colony forming unit assays (CFU) were performed independently in triplicates over a minimum of seven population doublings (UC-MSCs: passages 1–3, BM-MSCs: passages 2–4) examining all three medium types (A, B and C)
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Fig2: Experimental workflow: Isolation and characterization of umbilical cord (UC-) and bone marrow (BM-) MSCs. UC-MSCs group 1 (left) and BM-MSCs, group3 (right) were isolated using medium condition A exclusively. UC-MSCs group 2 (middle) were isolated in all three medium conditions (A, white; B, grey and C, black). After isolation and expansion, all MSC types tested by flow cytometric analysis and differentiation assays. Proliferation and colony forming unit assays (CFU) were performed independently in triplicates over a minimum of seven population doublings (UC-MSCs: passages 1–3, BM-MSCs: passages 2–4) examining all three medium types (A, B and C)

Mentions: UC-MSCs of five independent donations (group 1, Fig. 2) were initially isolated using standard medium A only. Another five donations (group 2) were immediately separated into three cord pieces and differentially isolated in either medium condition A, B or C. We further compared the functional response of UC-MSCs to various media types with BM-MSCs (group 3). Because we regularly collected BM-aspirates using heparin, we took BM-MSCs formerly isolated in standard medium A (corresponding to UC-MSCs group1) as controls. After expansion, all MSCs were characterized by flow cytometric analysis and differentiation assays as well as by proliferation and colony forming unit (CFU) assays over three subsequent passages (Fig. 2).Fig. 2


Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.

Laner-Plamberger S, Lener T, Schmid D, Streif DA, Salzer T, Öller M, Hauser-Kronberger C, Fischer T, Jacobs VR, Schallmoser K, Gimona M, Rohde E - J Transl Med (2015)

Experimental workflow: Isolation and characterization of umbilical cord (UC-) and bone marrow (BM-) MSCs. UC-MSCs group 1 (left) and BM-MSCs, group3 (right) were isolated using medium condition A exclusively. UC-MSCs group 2 (middle) were isolated in all three medium conditions (A, white; B, grey and C, black). After isolation and expansion, all MSC types tested by flow cytometric analysis and differentiation assays. Proliferation and colony forming unit assays (CFU) were performed independently in triplicates over a minimum of seven population doublings (UC-MSCs: passages 1–3, BM-MSCs: passages 2–4) examining all three medium types (A, B and C)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4641400&req=5

Fig2: Experimental workflow: Isolation and characterization of umbilical cord (UC-) and bone marrow (BM-) MSCs. UC-MSCs group 1 (left) and BM-MSCs, group3 (right) were isolated using medium condition A exclusively. UC-MSCs group 2 (middle) were isolated in all three medium conditions (A, white; B, grey and C, black). After isolation and expansion, all MSC types tested by flow cytometric analysis and differentiation assays. Proliferation and colony forming unit assays (CFU) were performed independently in triplicates over a minimum of seven population doublings (UC-MSCs: passages 1–3, BM-MSCs: passages 2–4) examining all three medium types (A, B and C)
Mentions: UC-MSCs of five independent donations (group 1, Fig. 2) were initially isolated using standard medium A only. Another five donations (group 2) were immediately separated into three cord pieces and differentially isolated in either medium condition A, B or C. We further compared the functional response of UC-MSCs to various media types with BM-MSCs (group 3). Because we regularly collected BM-aspirates using heparin, we took BM-MSCs formerly isolated in standard medium A (corresponding to UC-MSCs group1) as controls. After expansion, all MSCs were characterized by flow cytometric analysis and differentiation assays as well as by proliferation and colony forming unit (CFU) assays over three subsequent passages (Fig. 2).Fig. 2

Bottom Line: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained.Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium.Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. s.laner-plamberger@salk.at.

ABSTRACT

Background: Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin.

Methods: We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages.

Results: The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure.

Conclusions: Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

No MeSH data available.


Related in: MedlinePlus