Cell Tissue Bank (2014) 15:267–275 DOI 10.1007/s10561-014-9437-x
BRIEF COMMUNICATION
Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank Romain Laurent • Aure´lie Nallet Laurent Obert • Laurence Nicod Florelle Gindraux
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Received: 12 August 2013 / Accepted: 6 March 2014 / Published online: 15 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Human amniotic membrane (hAM) is known to have good potential to help the regeneration of tissue. It has been used for over 100 years in many medical disciplines because of its properties, namely a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. In order to use this ‘‘boosted membrane’’ as an advanced therapeutic medicinal product for bone repair, we aimed to observe the influence of tissue culture and/or
R. Laurent (&) L. Obert L. Nicod F. Gindraux Intervention, Innovation, Imagery, Engineering in Health (EA 4268), SFR FED 4234, University of Franche-Comte´, Besanc¸on, France e-mail: [email protected] R. Laurent Pediatric Surgery Service, University Hospital, Besanc¸on, France
cryopreservation on cell viability and tissue structure, and secondly, to adapt to a tissue bank, identify easy processes to store hAM containing viable cells and to verify the quality of the graft before its release for use. To this end, we tested different published culture or cryopreservation storage conditions and cell viability assays. Tissue structure was evaluated by Giemsa staining and was compared to histological analysis. Preliminary results show no dramatic decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data highlight that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Finally, we report that trypan blue and Giemsa staining could constitute controls that are ‘‘materially and easily transferable’’ to a tissue bank. Keywords Amniotic membrane Storage Viability Structure Qualification Tissue bank
A. Nallet Novotec, Lyon, France L. Obert F. Gindraux Orthopedic and Traumatology Surgery Service, University Hospital, Besanc¸on, France F. Gindraux Clinical Investigation Center in Biotherapy, University Hospital, Besanc¸on, France e-mail: [email protected]
The human placenta consists of the amniotic and chorionic fetal membranes and maternal cells, which are derived from maternal circulation and the endometrium. The amnion, also widely called the ‘‘amniotic membrane (AM)’’, comprises a single layer of ectodermally derived columnar epithelial cells adhered to a basement membrane largely composed of types I–VII
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collagen, elastin, laminin, and fibronectin, which in turn is attached to an underlying layer of connective tissue. The connective tissue has three components: an acellular compact layer composed of reticular fibers; a fibroblast layer, which is a loose reticular network containing sporadic fibroblasts; and a spongy layer which is in contact with the underlying chorion and consist of a complex network of fine fibrils surrounded by mucus (for references see: Wilshaw et al. (2006) and Riau et al. (2009)). The use of human amniotic membrane (hAM) has a history spanning more than 100 years. The first reported clinical use of hAM was in 1910, when it was applied in skin transplantation (Trelford and Trelford-Sauder 1979). Shortly after, its application was expanded to treat burned and ulcerated skin, and conjunctival defects. Since its rediscovery in 1995, it has been widely applied in ophthalmology, soft tissue surgery, and wound healing (Parolini et al. 2010; Mamede et al. 2012; Rennie et al. 2012). To date, hAM has clinical indication in ophthalmology and is being tested in clinical trials in dermatology and soft tissue surgery. Many beneficial properties of hAM, including anti-bacterial, anti-viral, anti-inflammatory, analgesic, anti-fibrotic, anatomical and vapor barrier properties have been reported and it reportedly facilitates wound healing, re-epithelialization and reduction of scarring (Kjaergaard et al. 1999; Li et al. 2005; Stock et al. 2007; Parolini et al. 2008; Tao and Fan 2009; Insausti et al. 2010). These pleiotropic functions are related in part to its capacity to synthesize and release biologically active substances, including cytokines and signaling molecules (Parolini et al. 2008; Insausti et al. 2010). Although fresh hAM (f-hAM) is frequently used (Trelford and Trelford-Sauder 1979), the potential risk of disease transmission should be taken into consideration. To overcome doubts about infectiousness and to respect the transplantation laws of many countries, several preserving techniques, such as cryopreservation, freeze-drying or gamma-sterilization have been developed over the years. Long-term storage of 6 months avoids the possibility of the donor being in the ‘‘window period’’ of infection. Questions remain as to how these different processing and preservation methods influence sterility, viability, growth factor release and re-epithelialization capacity (Kruse et al. 2000; Adds et al. 2001; Hennerbichler et al. 2007; von Versen-Hoynck et al. 2004; Parolini et al. 2008; von
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Versen-Hoeynck et al. 2008; Riau et al. 2009). Indeed, the literature regarding cell viability is sparse, and findings are contradictory, depending on the techniques used (Burgos and Faulk 1981; Kruse et al. 2000; Kubo et al. 2001; Adds et al. 2001; Wilshaw et al. 2006; Hennerbichler et al. 2007; Wolbank et al. 2009). For several years, our group has been working on bone repair at R&D and clinical levels (Deschaseaux et al. 2003; Obert et al. 2005; Gindraux et al. 2007, 2010; Masquelet and Obert 2010; Zappaterra et al. 2011; Zwetyenga et al. 2012; Houvet and Obert 2013) and we aimed to use intact hAM in this innovative indication and new market (Gindraux et al. 2013). Therefore, we studied, firstly, its osteogenic differentiation potential in in vitro studies, as previously reported by Lindenmair et al. (2010), and in an animal model. Secondly, based on previously reported data (Burgos and Faulk 1981; Kruse et al. 2000; Adds et al. 2001; Kubo et al. 2001; Wilshaw et al. 2006; Hennerbichler et al. 2007), we aimed to adapt to a tissue bank easy processes to store hAM containing viable cells able to osteodifferentiate, or viable osteodifferentiated hAM. Lastly, we aimed to verify the quality of this graft before its release for use. To this end, we tested different conditions of storage (tissue culture and/or cryopreservation) and different assays to evaluate cell viability and tissue structure. Human amniotic membrane were collected by a local tissue bank from consenting healthy mothers (tested seronegative for HIV, cytomegalovirus, Toxoplasma gondii, Hepatitis B and C virus, and syphilis) during routine Caesarean section births. Fetal membranes were separated from the placenta and kept sterile at 4 °C in 0.9 % saline and antibiotic solution. Within 24 h, hAM was peeled off the chorionic membrane by blunt dissection. Pieces of 5 cm in diameter were cut and put on sterile discs of nitrocellulose (Sartorius Stedim Biotech GmbH, Goettingen, Germany). At least 20 patches of f-hAM were obtained per placenta and underwent different treatments [tissue culture and/or cryopreservation with dimethyl-sulfoxide (DMSO) or glycerol] to observe the influence of these treatments on cell viability and tissue structure. Analysis were performed on: f-hAM; cryopreserved hAM (hereafter called ‘‘cryo-hAM’’); cultured hAM in standard medium (SM) (called ‘‘SM-hAM’’) or in osteogenic medium (OM) (called ‘‘OM-hAM’’) and finally on SM-hAM or OM-hAM
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undergoing cryopreservation (called ‘‘SM-cryo-hAM or OM-cryo-hAM’’); Three hAM were investigated and five patches were used per condition. For culture, pieces of f-hAM were removed from the nitrocellulose and cultured in Petri dishes for 3 weeks in either SM containing a-minimal essential medium (a-MEM, Gibco, Grand Island, USA), 10 % fetal bovine serum (Hyclone, Logan, USA), 1 % antibiotics, penicillin–streptomycin and used as control; or OM adapted from Pittenger et al. (1999) (called ‘‘pOM’’) and composed of SM supplemented by 2.16 mg/ml bglycerophosphate (Sigma–Aldrich, Saint Louis, USA), 50 lg/ml ascorbic acid (Sigma–Aldrich, Saint Louis, USA) and 20 lg/ml dexamethasone (Sigma–Aldrich, Saint Louis, USA); or commercial OM (StemPro
Osteogenesis Differentiation Kit, Gibco, Grand Island, USA) (called ‘‘cOM’’). For cryopreservation, f-hAM, SM-hAM or OMhAM put on the disc of nitrocellulose were directly frozen at -80 °C according to standard procedures established by the local tissue bank and validated by the French Health Products Safety Agency (Franck et al. 2000). The cryoconservation solutions were either composed of 50 % glycerol (Sigma–Aldrich, Saint Louis, USA) or 10 % DMSO (Sigma–Aldrich, Saint Louis, USA) in Roswell Park Memorial Institute medium (RPMI, Gibco, Grand Island, USA). Samples were kept frozen for 3 months and analyzed after thawing at room temperature in physiological saline baths.
Fig. 1 Trypan blue staining (a, b, c, d) performed on f-hAM (a, b) and cryo-hAM in Glycerol/RPMI (c, d). Macroscopic (a, c) and microscopic (b, d; scale bar 200 lm) observations showed an increase of dead cells after cryopreservation and thawing revealed by an increase in stain intensity (semiquantitative assessment). Calcein-AM/DAPI staining (e, f)
achieved on f-hAM (e) and OM-hAM (f) showing a destruction of epithelial layer in OM condition due to the mineralization (940). Macroscopic observations (g, h) of cultured SM-hAM (g) and in OM-hAM (h) for 3 weeks; mineralization was observed in OM condition and correspond to opacity of the tissue (h) compared to the control (g)
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Cell viability was assessed on f-hAM or treated hAM and was evaluated by trypan blue (Sigma–Aldrich, Saint Louis, USA) and calcein-acetoxy-methyl ester (Calcein-AM; Sigma–Aldrich, Saint Louis, USA) and 40 ,6diamidino-2-phenylindol (DAPI; Sigma–Aldrich, Saint Louis, USA) staining and with an EZ4U cell proliferation and cytotoxicity assay (Biomedica, Vienna, Austria) as previously described (Hennerbichler et al. 2007; Kubo et al. 2001; Lindenmair et al. 2010). Trypan blue and EZ4U assay were also performed on cells isolated from these tissues. For this, hAM were digested with 0.05 % trypsin/ethylenediaminetetraacetic acid (Biowest, Nuaille´, France) for 20 min and filtered on a 100 lm cell stainer to collected amniotic epithelial cells (AEC) after centrifugation. Undigested membranes were incubated with 2.4 U/ml dispase (BD Bioscience, Bedford, United Kingdom) for 10 min and then with 1 mg/ml collagenase A (Roche Diagnostics, Indianapolis, USA) for 3 h. After filtration on a 100 lm cell stainer and centrifugation, mesenchymal stromal cells (MSCs) were isolated and pooled with AEC. Tissue structure of f-AM or treated hAM was observed microscopically on whole Giemsa stained tissue (technique adapted from Castro-Malaspina et al. (1980)), and on 4 % formalin fixed, paraffin-embedded and sectioned tissue, stained by hematoxylin, eosin and saffron (HES). Alizarin red staining was used to detect calcium salts in tissue sections, highlighting features of mineralization. Tissue structure was also characterized by immunohistochemistry using specific antibodies: rabbit polyclonal antihuman collagen I (20121 Novotec, Lyon, France), anti-human collagen IV (20411, Novotec, Lyon, France), anti-human elastin (25011, Novotec, Lyon, France), mouse monoclonal anti-alpha smooth muscle actin (M0851, Dako, Glostrup, Denmark), mouse monoclonal anti-human CD44 (E17360, Spring Biosciences, Pleasanton, USA) and rabbit monoclonal anti-human CD73 (5362-1, Epitomics, Burlingame, USA). Antigen–antibody complexes were revealed by peroxydase conjugates antibody Dako HRP Envision (K4000/K4002, Dako, Glostrup, Denmark). Cell viability tests performed on f-hAM and treated hAM are presented in Figs. 1, 2. Trypan blue staining performed on f-hAM (Fig. 1a, b) and cryo-hAM in Glycerol/RPMI (Fig. 1c, d) showed, according to semi-quantitative assessment performed by two independent operators, differences in staining, highlighting a high percentage of dead cells when hAM is
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cryopreserved (Fig. 1c, d). This procedure is the same procedure that is used to evaluate the viability of cells in corneas in many eye tissue banks (Rodrigues et al. 2009). Experts from our tissue bank who are trained in the analysis of viability in corneas also analyzed our samples and gave their independent conclusions. Calcein-AM/DAPI staining performed on f-hAM (Fig. 1e) and OM-hAM (Fig. 1f) showed destruction of the epithelial layer when hAM was cultured in OM and this was confirmed by macroscopic observations (Fig. 1g, h) and by microscopic observation with alizarin red staining (Fig. 4o, r) which showed mineralization in osteogenic condition (Fig. 1h, r) compared to control, cultured SM-hAM (Figs. 1g, 4o). This mineralization corresponded visually to opacity, whereas cultured SM-hAM remained transparent (like the f-hAM). All these results were correlated to EZ4U assays (Fig. 2), which showed a significant 60 % decrease in cell viability relative to f-hAM after 3 weeks of culture in both SM (SM-hAM) and OM (pOM-hAM and cOM-hAM). Similar results in the literature reported a decrease of 40–65 % after 4 weeks of culture, without any significant difference between SM and OM (Lindenmair et al. 2010).
Fig. 2 Cell viability of f-hAM; cultured hAM in standard medium (SM-hAM) or in osteogenic medium (adapted from Pittenger (pOM-hAM) or commercial OM (cOM-hAM)) for 3 weeks; cryopreserved hAM (cryo-hAM); and SM-hAM or OM-hAM undergoing cryopreservation (SM-cryo-hAM or OMcryo-hAM) for 3 months in DMSO/RPMI (n = 4). Data presented are mean ± SD of % viability relative to f-hAM
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Moreover, our results showed that cryopreservation (cryo-hAM) affects cell viability to the same extent as observed by other authors (Hennerbichler et al. 2007). No additional decrease due to cryoconservation was observed when f-hAM was cultured in SM (SM-cryohAM) or in OM (cOM-cryo-hAM) before freezing, and
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in this case, the decrease in viable cells relative to f-hAM was about 60 %. With this EZ4U assay, according to standard deviation, we were unable to note a significant difference between f-hAM and treated hAM (SM/pOM/cOM-hAM; cryo-hAM and SM/cOM-cryo-hAM). The EZ4U test performed on
Fig. 3 Giemsa staining performed on f-hAM (a, b, c, d) and cryo-hAM in Glycerol/RPMI (e, f, g, h). Epithelium (a, c, e, g) and fibroblast layer (b, d, f, h). Microscopic observations [scale bar 33 lm (a, b); 66 lm (e, f); 100 lm (c, d); 200 lm (g, h)]
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Cell Tissue Bank (2014) 15:267–275 b Fig. 4 Histological staining and immunolabelings of f-hAM (a,
b, c, d, e, f), cryo-hAM in Glycerol/RPMI (g, h, i, j, k, l), SMhAM (m, n, o) and OM-hAM (p, q, r). a, g, m, p HES staining, b, h anti-human CD44,E17360, Spring Biosciences, c, i antihuman CD73, 5362-1, Epitomics, d, j, n, q anti-human type I collagen, 20121, Novotec, e, k anti-human type IV collagen, 20411, Novotec, f, l anti-human elastin, 25011, Novotec, (o, r) alizarin red staining. Microscopic observations (scale bar 50 lm)
cells isolated from f-hAM cultured 3 weeks in SM, Pittenger adapted OM and commercial OM showed a significant decrease in viability, with viability of 41, 40 and 50 % respectively, whereas trypan blue staining did not reveal dead cells. Indeed, cells from the same population were able to grow in SM and to mineralize in OM (data not shown). Same observations were founded for cryo-hAM (in DMSO) suggesting unfortunately, in this preliminary study, a no-correlation between EZ4U and trypan blue staining. Thus, our first results suggest that tissue culturing in SM or OM did not dramatically alter cell viability and function because cells were capable of growing and mineralizing. The same can be said of cryoconservation in DMSO, making our analysis difficult to interpret in the light of the limited literature on the subject (Burgos and Faulk 1981; Dohrmann et al. 1990; Kruse et al. 2000; Rama et al. 2001; Kubo et al. 2001). Cell viability and function are very important parameters, because they are directly linked to the use of hAM in regenerative medicine. Tissue banks in France and other countries carry out virological and bacteriological controls to authorize the release of hAM. It should be noted that up to now, cell viability has not been taken into consideration for clinical use of hAM in ophthalmology. There is even ongoing debate as to whether cryo-hAM contains viable cells or not. Literature regarding evaluation of cell viability is very poor and often involved long techniques that require many specific materials (Burgos and Faulk 1981; Kruse et al. 2000; Kubo et al. 2001; Adds et al. 2001; Wilshaw et al. 2006; Hennerbichler et al. 2007). We purport that trypan blue staining on intact tissue, already used for cornea grafts (Rodrigues et al. 2009), could be easily improved and implemented in a tissue bank. In order to develop an easy technique to observe tissue structure, we adapted to intact hAM a staining technique used to observed MSCs or fibroblastic colony unit forming cells in bone marrow cultures (Castro-Malaspina et al. 1980). Giemsa staining
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performed on f-hAM (Fig. 3a–d) and cryo-hAM (Fig. 3e–h) showed that the epithelium fascia in f-hAM is a well-organized structure with regular cuboidal cells embedded in a dense and homogeneous extracellular matrix (Fig. 3a–c). At a greater microscopic field depth, fibroblast-like cells were observed (Fig. 3b–d). On the contrary, in the cryo-hAM, the integrity of the tissue was not preserved, as indicated by the damaged cuboid cells embedded in a heterogeneous loose and fractured matrix (Fig. 3e–g). Similar observations with damaged fibroblast-like cells were made with the deep microscopic field (Fig. 3f, h). These results were compared to histological staining and immunolabeling (Fig. 4a–n), which confirmed both cell and matrix damage in cryo-hAM (Fig. 4g–l) as compared to f-hAM (Fig. 4a–f). In particular, a decrease in cellular markers (Fig. 4h, i), alteration of collagen I and elastin fibers (Fig. 4j, l) and shrinkage of basement membrane (Fig. 4k) was observed, whereas integrity of f-hAM tissue was well preserved (Fig. 4a–f). Membranes cultivated for 3 weeks also showed severe damage, in SM (Fig. 4m–o) as well as in OM (Fig. 4p–r), namely alteration of cell morphology with a small loss of epithelial cells (Fig. 4m, p) and matrix degradation with loss of fiber structure (Fig. 4n, q). Results of alizarin red staining confirmed the mineralization of OM-hAM presumed on macroscopical observations (Fig. 4o, r). Cell viability, which we plan to evaluate by propidium iodide staining in another study, is required to appraise the extent of cell damage. Although Kruse et al. (2000) did not show any epithelial cell border modification with cryopreservation, our detailed immunolabeling results show that protein expression seems to be significantly modified with culture and/or cryopreservation. Stadler et al. (2008) reported phenotypic alterations of human AEC during in vitro cultivation, which might be responsible for a functional reduction of the differentiation potential. Therefore, it is noteworthy that tissue damage caused by either tissue culture or cryopreservation may possible affect the functional abilities of the hAM and its use in regenerative medicine. Tissue structure damage observed with detailed immunolabeling performed on treated hAM is not sufficiently described in the literature (Kubo et al. 2001; von Versen-Hoynck et al. 2004; Wilshaw et al. 2006; von Versen-Hoeynck et al. 2008; Riau et al. 2009; Guo et al. 2012) and further research building on our histological
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observations could help to identify the real potential of treated hAM for tissue repair. To prevent cell viability decrease and/or tissue structure damage, we purport that one solution would be to avoid culture and/or preservation of hAM altogether. With the introduction of nucleic acid testing in tissue banks (Morel 2011), the ‘‘window period’’ for infection of 6 months can be shorted to a few days (\7 days). In this case, f-hAM could be used without the risks of culture and/or cryopreservation that can influence cell viability, tissue structure and involve phenotypic shift resulting in loss of function (Stadler et al. 2008). Further, these preservation techniques are time consuming and costly, with a risk of contamination with pathogens, and present difficulties gaining market authorization by competent authorities (Adds et al. 2001; von Versen-Hoeynck et al. 2008). In this case, hAM could be stored as currently done for the cornea, and new conservation medium could be developed for this purpose and even patented, as for the patents of Zhou (2010) and Tong (2012). We are also investigating a dynamic culture instead of a static culture to improve medium penetration. Finally, we suggest that trypan blue and Giemsa staining would be a useful addition to the arsenal of quality controls necessary for the release of grafts. In our opinion, these techniques would be easy to implement to a tissue bank. Acknowledgments Thanks to the local tissue bank ‘‘Cell and tissue engineering activities’’ from French blood transfusion center ‘‘Bourgogne Franche-Comte´’’, Besanc¸on, France for amniotic membranes collection. Thanks also to Martine Melin and Daniel Hartmann (Novotec, Lyon, France) for histological studies and scientific advice. The authors also thank Fiona Ecarnot (EA3920, University Hospital Besancon, France) for editorial assistance. This work was supported by the Foundation of Transplantation (FDTSFV), Saint Apollinaire, France. Conflict of interests conflicts of interest.
The authors confirm that there are no
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BRIEF COMMUNICATION
Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank Romain Laurent • Aure´lie Nallet Laurent Obert • Laurence Nicod Florelle Gindraux
• •
Received: 12 August 2013 / Accepted: 6 March 2014 / Published online: 15 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Human amniotic membrane (hAM) is known to have good potential to help the regeneration of tissue. It has been used for over 100 years in many medical disciplines because of its properties, namely a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. In order to use this ‘‘boosted membrane’’ as an advanced therapeutic medicinal product for bone repair, we aimed to observe the influence of tissue culture and/or
R. Laurent (&) L. Obert L. Nicod F. Gindraux Intervention, Innovation, Imagery, Engineering in Health (EA 4268), SFR FED 4234, University of Franche-Comte´, Besanc¸on, France e-mail: [email protected] R. Laurent Pediatric Surgery Service, University Hospital, Besanc¸on, France
cryopreservation on cell viability and tissue structure, and secondly, to adapt to a tissue bank, identify easy processes to store hAM containing viable cells and to verify the quality of the graft before its release for use. To this end, we tested different published culture or cryopreservation storage conditions and cell viability assays. Tissue structure was evaluated by Giemsa staining and was compared to histological analysis. Preliminary results show no dramatic decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data highlight that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Finally, we report that trypan blue and Giemsa staining could constitute controls that are ‘‘materially and easily transferable’’ to a tissue bank. Keywords Amniotic membrane Storage Viability Structure Qualification Tissue bank
A. Nallet Novotec, Lyon, France L. Obert F. Gindraux Orthopedic and Traumatology Surgery Service, University Hospital, Besanc¸on, France F. Gindraux Clinical Investigation Center in Biotherapy, University Hospital, Besanc¸on, France e-mail: [email protected]
The human placenta consists of the amniotic and chorionic fetal membranes and maternal cells, which are derived from maternal circulation and the endometrium. The amnion, also widely called the ‘‘amniotic membrane (AM)’’, comprises a single layer of ectodermally derived columnar epithelial cells adhered to a basement membrane largely composed of types I–VII
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collagen, elastin, laminin, and fibronectin, which in turn is attached to an underlying layer of connective tissue. The connective tissue has three components: an acellular compact layer composed of reticular fibers; a fibroblast layer, which is a loose reticular network containing sporadic fibroblasts; and a spongy layer which is in contact with the underlying chorion and consist of a complex network of fine fibrils surrounded by mucus (for references see: Wilshaw et al. (2006) and Riau et al. (2009)). The use of human amniotic membrane (hAM) has a history spanning more than 100 years. The first reported clinical use of hAM was in 1910, when it was applied in skin transplantation (Trelford and Trelford-Sauder 1979). Shortly after, its application was expanded to treat burned and ulcerated skin, and conjunctival defects. Since its rediscovery in 1995, it has been widely applied in ophthalmology, soft tissue surgery, and wound healing (Parolini et al. 2010; Mamede et al. 2012; Rennie et al. 2012). To date, hAM has clinical indication in ophthalmology and is being tested in clinical trials in dermatology and soft tissue surgery. Many beneficial properties of hAM, including anti-bacterial, anti-viral, anti-inflammatory, analgesic, anti-fibrotic, anatomical and vapor barrier properties have been reported and it reportedly facilitates wound healing, re-epithelialization and reduction of scarring (Kjaergaard et al. 1999; Li et al. 2005; Stock et al. 2007; Parolini et al. 2008; Tao and Fan 2009; Insausti et al. 2010). These pleiotropic functions are related in part to its capacity to synthesize and release biologically active substances, including cytokines and signaling molecules (Parolini et al. 2008; Insausti et al. 2010). Although fresh hAM (f-hAM) is frequently used (Trelford and Trelford-Sauder 1979), the potential risk of disease transmission should be taken into consideration. To overcome doubts about infectiousness and to respect the transplantation laws of many countries, several preserving techniques, such as cryopreservation, freeze-drying or gamma-sterilization have been developed over the years. Long-term storage of 6 months avoids the possibility of the donor being in the ‘‘window period’’ of infection. Questions remain as to how these different processing and preservation methods influence sterility, viability, growth factor release and re-epithelialization capacity (Kruse et al. 2000; Adds et al. 2001; Hennerbichler et al. 2007; von Versen-Hoynck et al. 2004; Parolini et al. 2008; von
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Versen-Hoeynck et al. 2008; Riau et al. 2009). Indeed, the literature regarding cell viability is sparse, and findings are contradictory, depending on the techniques used (Burgos and Faulk 1981; Kruse et al. 2000; Kubo et al. 2001; Adds et al. 2001; Wilshaw et al. 2006; Hennerbichler et al. 2007; Wolbank et al. 2009). For several years, our group has been working on bone repair at R&D and clinical levels (Deschaseaux et al. 2003; Obert et al. 2005; Gindraux et al. 2007, 2010; Masquelet and Obert 2010; Zappaterra et al. 2011; Zwetyenga et al. 2012; Houvet and Obert 2013) and we aimed to use intact hAM in this innovative indication and new market (Gindraux et al. 2013). Therefore, we studied, firstly, its osteogenic differentiation potential in in vitro studies, as previously reported by Lindenmair et al. (2010), and in an animal model. Secondly, based on previously reported data (Burgos and Faulk 1981; Kruse et al. 2000; Adds et al. 2001; Kubo et al. 2001; Wilshaw et al. 2006; Hennerbichler et al. 2007), we aimed to adapt to a tissue bank easy processes to store hAM containing viable cells able to osteodifferentiate, or viable osteodifferentiated hAM. Lastly, we aimed to verify the quality of this graft before its release for use. To this end, we tested different conditions of storage (tissue culture and/or cryopreservation) and different assays to evaluate cell viability and tissue structure. Human amniotic membrane were collected by a local tissue bank from consenting healthy mothers (tested seronegative for HIV, cytomegalovirus, Toxoplasma gondii, Hepatitis B and C virus, and syphilis) during routine Caesarean section births. Fetal membranes were separated from the placenta and kept sterile at 4 °C in 0.9 % saline and antibiotic solution. Within 24 h, hAM was peeled off the chorionic membrane by blunt dissection. Pieces of 5 cm in diameter were cut and put on sterile discs of nitrocellulose (Sartorius Stedim Biotech GmbH, Goettingen, Germany). At least 20 patches of f-hAM were obtained per placenta and underwent different treatments [tissue culture and/or cryopreservation with dimethyl-sulfoxide (DMSO) or glycerol] to observe the influence of these treatments on cell viability and tissue structure. Analysis were performed on: f-hAM; cryopreserved hAM (hereafter called ‘‘cryo-hAM’’); cultured hAM in standard medium (SM) (called ‘‘SM-hAM’’) or in osteogenic medium (OM) (called ‘‘OM-hAM’’) and finally on SM-hAM or OM-hAM
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undergoing cryopreservation (called ‘‘SM-cryo-hAM or OM-cryo-hAM’’); Three hAM were investigated and five patches were used per condition. For culture, pieces of f-hAM were removed from the nitrocellulose and cultured in Petri dishes for 3 weeks in either SM containing a-minimal essential medium (a-MEM, Gibco, Grand Island, USA), 10 % fetal bovine serum (Hyclone, Logan, USA), 1 % antibiotics, penicillin–streptomycin and used as control; or OM adapted from Pittenger et al. (1999) (called ‘‘pOM’’) and composed of SM supplemented by 2.16 mg/ml bglycerophosphate (Sigma–Aldrich, Saint Louis, USA), 50 lg/ml ascorbic acid (Sigma–Aldrich, Saint Louis, USA) and 20 lg/ml dexamethasone (Sigma–Aldrich, Saint Louis, USA); or commercial OM (StemPro
Osteogenesis Differentiation Kit, Gibco, Grand Island, USA) (called ‘‘cOM’’). For cryopreservation, f-hAM, SM-hAM or OMhAM put on the disc of nitrocellulose were directly frozen at -80 °C according to standard procedures established by the local tissue bank and validated by the French Health Products Safety Agency (Franck et al. 2000). The cryoconservation solutions were either composed of 50 % glycerol (Sigma–Aldrich, Saint Louis, USA) or 10 % DMSO (Sigma–Aldrich, Saint Louis, USA) in Roswell Park Memorial Institute medium (RPMI, Gibco, Grand Island, USA). Samples were kept frozen for 3 months and analyzed after thawing at room temperature in physiological saline baths.
Fig. 1 Trypan blue staining (a, b, c, d) performed on f-hAM (a, b) and cryo-hAM in Glycerol/RPMI (c, d). Macroscopic (a, c) and microscopic (b, d; scale bar 200 lm) observations showed an increase of dead cells after cryopreservation and thawing revealed by an increase in stain intensity (semiquantitative assessment). Calcein-AM/DAPI staining (e, f)
achieved on f-hAM (e) and OM-hAM (f) showing a destruction of epithelial layer in OM condition due to the mineralization (940). Macroscopic observations (g, h) of cultured SM-hAM (g) and in OM-hAM (h) for 3 weeks; mineralization was observed in OM condition and correspond to opacity of the tissue (h) compared to the control (g)
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Cell viability was assessed on f-hAM or treated hAM and was evaluated by trypan blue (Sigma–Aldrich, Saint Louis, USA) and calcein-acetoxy-methyl ester (Calcein-AM; Sigma–Aldrich, Saint Louis, USA) and 40 ,6diamidino-2-phenylindol (DAPI; Sigma–Aldrich, Saint Louis, USA) staining and with an EZ4U cell proliferation and cytotoxicity assay (Biomedica, Vienna, Austria) as previously described (Hennerbichler et al. 2007; Kubo et al. 2001; Lindenmair et al. 2010). Trypan blue and EZ4U assay were also performed on cells isolated from these tissues. For this, hAM were digested with 0.05 % trypsin/ethylenediaminetetraacetic acid (Biowest, Nuaille´, France) for 20 min and filtered on a 100 lm cell stainer to collected amniotic epithelial cells (AEC) after centrifugation. Undigested membranes were incubated with 2.4 U/ml dispase (BD Bioscience, Bedford, United Kingdom) for 10 min and then with 1 mg/ml collagenase A (Roche Diagnostics, Indianapolis, USA) for 3 h. After filtration on a 100 lm cell stainer and centrifugation, mesenchymal stromal cells (MSCs) were isolated and pooled with AEC. Tissue structure of f-AM or treated hAM was observed microscopically on whole Giemsa stained tissue (technique adapted from Castro-Malaspina et al. (1980)), and on 4 % formalin fixed, paraffin-embedded and sectioned tissue, stained by hematoxylin, eosin and saffron (HES). Alizarin red staining was used to detect calcium salts in tissue sections, highlighting features of mineralization. Tissue structure was also characterized by immunohistochemistry using specific antibodies: rabbit polyclonal antihuman collagen I (20121 Novotec, Lyon, France), anti-human collagen IV (20411, Novotec, Lyon, France), anti-human elastin (25011, Novotec, Lyon, France), mouse monoclonal anti-alpha smooth muscle actin (M0851, Dako, Glostrup, Denmark), mouse monoclonal anti-human CD44 (E17360, Spring Biosciences, Pleasanton, USA) and rabbit monoclonal anti-human CD73 (5362-1, Epitomics, Burlingame, USA). Antigen–antibody complexes were revealed by peroxydase conjugates antibody Dako HRP Envision (K4000/K4002, Dako, Glostrup, Denmark). Cell viability tests performed on f-hAM and treated hAM are presented in Figs. 1, 2. Trypan blue staining performed on f-hAM (Fig. 1a, b) and cryo-hAM in Glycerol/RPMI (Fig. 1c, d) showed, according to semi-quantitative assessment performed by two independent operators, differences in staining, highlighting a high percentage of dead cells when hAM is
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cryopreserved (Fig. 1c, d). This procedure is the same procedure that is used to evaluate the viability of cells in corneas in many eye tissue banks (Rodrigues et al. 2009). Experts from our tissue bank who are trained in the analysis of viability in corneas also analyzed our samples and gave their independent conclusions. Calcein-AM/DAPI staining performed on f-hAM (Fig. 1e) and OM-hAM (Fig. 1f) showed destruction of the epithelial layer when hAM was cultured in OM and this was confirmed by macroscopic observations (Fig. 1g, h) and by microscopic observation with alizarin red staining (Fig. 4o, r) which showed mineralization in osteogenic condition (Fig. 1h, r) compared to control, cultured SM-hAM (Figs. 1g, 4o). This mineralization corresponded visually to opacity, whereas cultured SM-hAM remained transparent (like the f-hAM). All these results were correlated to EZ4U assays (Fig. 2), which showed a significant 60 % decrease in cell viability relative to f-hAM after 3 weeks of culture in both SM (SM-hAM) and OM (pOM-hAM and cOM-hAM). Similar results in the literature reported a decrease of 40–65 % after 4 weeks of culture, without any significant difference between SM and OM (Lindenmair et al. 2010).
Fig. 2 Cell viability of f-hAM; cultured hAM in standard medium (SM-hAM) or in osteogenic medium (adapted from Pittenger (pOM-hAM) or commercial OM (cOM-hAM)) for 3 weeks; cryopreserved hAM (cryo-hAM); and SM-hAM or OM-hAM undergoing cryopreservation (SM-cryo-hAM or OMcryo-hAM) for 3 months in DMSO/RPMI (n = 4). Data presented are mean ± SD of % viability relative to f-hAM
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Moreover, our results showed that cryopreservation (cryo-hAM) affects cell viability to the same extent as observed by other authors (Hennerbichler et al. 2007). No additional decrease due to cryoconservation was observed when f-hAM was cultured in SM (SM-cryohAM) or in OM (cOM-cryo-hAM) before freezing, and
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in this case, the decrease in viable cells relative to f-hAM was about 60 %. With this EZ4U assay, according to standard deviation, we were unable to note a significant difference between f-hAM and treated hAM (SM/pOM/cOM-hAM; cryo-hAM and SM/cOM-cryo-hAM). The EZ4U test performed on
Fig. 3 Giemsa staining performed on f-hAM (a, b, c, d) and cryo-hAM in Glycerol/RPMI (e, f, g, h). Epithelium (a, c, e, g) and fibroblast layer (b, d, f, h). Microscopic observations [scale bar 33 lm (a, b); 66 lm (e, f); 100 lm (c, d); 200 lm (g, h)]
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Cell Tissue Bank (2014) 15:267–275 b Fig. 4 Histological staining and immunolabelings of f-hAM (a,
b, c, d, e, f), cryo-hAM in Glycerol/RPMI (g, h, i, j, k, l), SMhAM (m, n, o) and OM-hAM (p, q, r). a, g, m, p HES staining, b, h anti-human CD44,E17360, Spring Biosciences, c, i antihuman CD73, 5362-1, Epitomics, d, j, n, q anti-human type I collagen, 20121, Novotec, e, k anti-human type IV collagen, 20411, Novotec, f, l anti-human elastin, 25011, Novotec, (o, r) alizarin red staining. Microscopic observations (scale bar 50 lm)
cells isolated from f-hAM cultured 3 weeks in SM, Pittenger adapted OM and commercial OM showed a significant decrease in viability, with viability of 41, 40 and 50 % respectively, whereas trypan blue staining did not reveal dead cells. Indeed, cells from the same population were able to grow in SM and to mineralize in OM (data not shown). Same observations were founded for cryo-hAM (in DMSO) suggesting unfortunately, in this preliminary study, a no-correlation between EZ4U and trypan blue staining. Thus, our first results suggest that tissue culturing in SM or OM did not dramatically alter cell viability and function because cells were capable of growing and mineralizing. The same can be said of cryoconservation in DMSO, making our analysis difficult to interpret in the light of the limited literature on the subject (Burgos and Faulk 1981; Dohrmann et al. 1990; Kruse et al. 2000; Rama et al. 2001; Kubo et al. 2001). Cell viability and function are very important parameters, because they are directly linked to the use of hAM in regenerative medicine. Tissue banks in France and other countries carry out virological and bacteriological controls to authorize the release of hAM. It should be noted that up to now, cell viability has not been taken into consideration for clinical use of hAM in ophthalmology. There is even ongoing debate as to whether cryo-hAM contains viable cells or not. Literature regarding evaluation of cell viability is very poor and often involved long techniques that require many specific materials (Burgos and Faulk 1981; Kruse et al. 2000; Kubo et al. 2001; Adds et al. 2001; Wilshaw et al. 2006; Hennerbichler et al. 2007). We purport that trypan blue staining on intact tissue, already used for cornea grafts (Rodrigues et al. 2009), could be easily improved and implemented in a tissue bank. In order to develop an easy technique to observe tissue structure, we adapted to intact hAM a staining technique used to observed MSCs or fibroblastic colony unit forming cells in bone marrow cultures (Castro-Malaspina et al. 1980). Giemsa staining
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performed on f-hAM (Fig. 3a–d) and cryo-hAM (Fig. 3e–h) showed that the epithelium fascia in f-hAM is a well-organized structure with regular cuboidal cells embedded in a dense and homogeneous extracellular matrix (Fig. 3a–c). At a greater microscopic field depth, fibroblast-like cells were observed (Fig. 3b–d). On the contrary, in the cryo-hAM, the integrity of the tissue was not preserved, as indicated by the damaged cuboid cells embedded in a heterogeneous loose and fractured matrix (Fig. 3e–g). Similar observations with damaged fibroblast-like cells were made with the deep microscopic field (Fig. 3f, h). These results were compared to histological staining and immunolabeling (Fig. 4a–n), which confirmed both cell and matrix damage in cryo-hAM (Fig. 4g–l) as compared to f-hAM (Fig. 4a–f). In particular, a decrease in cellular markers (Fig. 4h, i), alteration of collagen I and elastin fibers (Fig. 4j, l) and shrinkage of basement membrane (Fig. 4k) was observed, whereas integrity of f-hAM tissue was well preserved (Fig. 4a–f). Membranes cultivated for 3 weeks also showed severe damage, in SM (Fig. 4m–o) as well as in OM (Fig. 4p–r), namely alteration of cell morphology with a small loss of epithelial cells (Fig. 4m, p) and matrix degradation with loss of fiber structure (Fig. 4n, q). Results of alizarin red staining confirmed the mineralization of OM-hAM presumed on macroscopical observations (Fig. 4o, r). Cell viability, which we plan to evaluate by propidium iodide staining in another study, is required to appraise the extent of cell damage. Although Kruse et al. (2000) did not show any epithelial cell border modification with cryopreservation, our detailed immunolabeling results show that protein expression seems to be significantly modified with culture and/or cryopreservation. Stadler et al. (2008) reported phenotypic alterations of human AEC during in vitro cultivation, which might be responsible for a functional reduction of the differentiation potential. Therefore, it is noteworthy that tissue damage caused by either tissue culture or cryopreservation may possible affect the functional abilities of the hAM and its use in regenerative medicine. Tissue structure damage observed with detailed immunolabeling performed on treated hAM is not sufficiently described in the literature (Kubo et al. 2001; von Versen-Hoynck et al. 2004; Wilshaw et al. 2006; von Versen-Hoeynck et al. 2008; Riau et al. 2009; Guo et al. 2012) and further research building on our histological
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observations could help to identify the real potential of treated hAM for tissue repair. To prevent cell viability decrease and/or tissue structure damage, we purport that one solution would be to avoid culture and/or preservation of hAM altogether. With the introduction of nucleic acid testing in tissue banks (Morel 2011), the ‘‘window period’’ for infection of 6 months can be shorted to a few days (\7 days). In this case, f-hAM could be used without the risks of culture and/or cryopreservation that can influence cell viability, tissue structure and involve phenotypic shift resulting in loss of function (Stadler et al. 2008). Further, these preservation techniques are time consuming and costly, with a risk of contamination with pathogens, and present difficulties gaining market authorization by competent authorities (Adds et al. 2001; von Versen-Hoeynck et al. 2008). In this case, hAM could be stored as currently done for the cornea, and new conservation medium could be developed for this purpose and even patented, as for the patents of Zhou (2010) and Tong (2012). We are also investigating a dynamic culture instead of a static culture to improve medium penetration. Finally, we suggest that trypan blue and Giemsa staining would be a useful addition to the arsenal of quality controls necessary for the release of grafts. In our opinion, these techniques would be easy to implement to a tissue bank. Acknowledgments Thanks to the local tissue bank ‘‘Cell and tissue engineering activities’’ from French blood transfusion center ‘‘Bourgogne Franche-Comte´’’, Besanc¸on, France for amniotic membranes collection. Thanks also to Martine Melin and Daniel Hartmann (Novotec, Lyon, France) for histological studies and scientific advice. The authors also thank Fiona Ecarnot (EA3920, University Hospital Besancon, France) for editorial assistance. This work was supported by the Foundation of Transplantation (FDTSFV), Saint Apollinaire, France. Conflict of interests conflicts of interest.
The authors confirm that there are no
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