Human platelet lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review.

BACKGROUND: Foetal bovine serum (FBS), is the most commonly used culture medium additive for in vitro cultures, despite its undefined composition, its potential immunogenicity and possible prion/zoonotic transmission. For these reasons, significant efforts have been targeted at finding a substitute, such as serum free-media or human platelet-lysates (hPL). Our aim is to critically appraise the state-of-art for hPL in the published literature, comparing its impact with FBS. MATERIALS AND METHODS: In June 2019 a systematic search of the entire Web of Science, Medline and PubMed database was performed with the following search terms: (mesenchymal stem cells) AND (fetal bovine serum OR fetal bovine calf) AND (human platelet lysate). Excluded from this search were review articles that were published before 2005, manuscripts in which mesenchymal stem cells (MSCs) were not from human sources, and when the FBS controls were missing. RESULTS: Based on our search algorithm, 56 papers were selected. A review of these papers indicated that hMSCs cultured with hPL showed a spindle-shaped elongated morphology, had higher proliferation indexes, similar cluster of differentiation (CD) markers and no significant variation in differentiation lineage (osteocyte, adipocyte, and chondrocyte) compared to those cultured with FBS. Main sources of primary hMSCs were either fat tissue or bone marrow; in a few studies cells isolated from alternative sources showed no relevant difference in their response. CONCLUSION: Despite the difference in medium choice and a lack of standardization of hPL manufacturing, the majority of publications support that hPL was at least as effective as FBS in promoting adhesion, survival and proliferation of hMSCs. We conclude that hPL should be considered a viable alternative to FBS in hMSCs culture-especially with a view for their clinical use.

Anatomically accurate 3D modelling and printing in a case of obstetric brachial plexus injury.

Obstetric brachial plexus injury is reported in 0.42 per 1000 births in UK and Ireland and are associated with a reduction in quality of life for the patient and their carers. In this report we describe the first use of a patient specific, anatomically accurate 3D model as a communication tool in the treatment of a complex case of posterior shoulder subluxation secondary to glenohumeral deformity resulting from obstetric brachial plexus injury. The use of 3D models for surgical planning is associated with decreased operating time and reduction of intra-operative blood loss, whilst their use in patient education increases patient understanding. In this case all surgeons surveyed agreed that it was useful and will use 3D modelling to improve consent processes and to conceptualise novel techniques for complex cases in future. This highly reproducible, low cost technique may be adapted to a variety of upper limb reconstructive surgeries, and as the resolution of image acquisition and additive manufacturing capabilities increase so too do the potential applications of this precise 3D printed surgical adjunct.

Morphological and microarray analysis of human fibroblasts cultured on nanocolumns produced by colloidal lithography.

The environment around a cell during in vitro culture is unlikely to mimic those in vivo. Preliminary experiments with nanotopography have shown that nanoscale features can strongly influence cell morphology, adhesion, proliferation and gene regulation, but the mechanisms mediating this cell response remain unclear. In this study a well defined nanotopography, consisting of 100 nm wide and 160 nm high cylindrical columns, was used in fibroblast culture. In order to build on previously published morphological data that showed changes in cell spreading on the nanocolumns, in this study gene regulation was monitored using a 1718 gene microarray. Transmission electron microscopy, fluorescent observation of actin and Rac and area quantification have been used to re-affirm the microarray observations. The results indicate that changes in cell spreading correlate with a number of gene up- and down-regulations as will be described within the manuscript.

Dynamic acoustic field activated cell separation (DAFACS).

Advances in diagnostics, cell and stem cell technologies drive the development of application-specific tools for cell and particle separation. Acoustic micro-particle separation offers a promising avenue for high-throughput, label-free, high recovery, cell and particle separation and isolation in regenerative medicine. Here, we demonstrate a novel approach utilizing a dynamic acoustic field that is capable of separating an arbitrary size range of cells. We first demonstrate the method for the separation of particles with different diameters between 6 and 45 µm and secondly particles of different densities in a heterogeneous medium. The dynamic acoustic field is then used to separate dorsal root ganglion cells. The shearless, label-free and low damage characteristics make this method of manipulation particularly suited for biological applications. Advantages of using a dynamic acoustic field for the separation of cells include its inherent safety and biocompatibility, the possibility to operate over large distances (centimetres), high purity (ratio of particle population, up to 100%), and high efficiency (ratio of separated particles over total number of particles to separate, up to 100%).

In vitro reaction of endothelial cells to polymer demixed nanotopography.

The introduction of topography to material surfaces has been shown to strongly affect cell behaviour, and the effects of micrometric surface morphologies have been extensively characterised. Research is now starting to investigate the reaction of cells to nanometric topography. This study used polymer demixing of polystyrene and poly(4-bromostyrene) producing nanometrically high islands, and observed endothelial cell response to the islands. Three island heights were investigated; these were 13, 35 and 95 nm. The cells were seen to be more spread on the manufactured topographies than that on flat surfaces of similar chemistry. Other morphological differences were also noted by histology, fluorescence and scanning electron microscopy, with many arcuate cells noted on the test surfaces, and cytoskeletal alignment along the arcuate features. Of the nanotopographies, the 13 nm islands were seen to give the largest response, with highly spread cell morphologies containing well-defined cytoskeleton.

Rapid fibroblast adhesion to 27nm high polymer demixed nano-topography.

It is well known that many cell types react strongly to micro-topography. It is rapidly becoming clear than cells will also react to nano-topography. Polymer demixing is a rapid and low-cost chemical method of producing nano-topography. This manuscript investigates human fibroblast response to 27nm high nano-islands produced by polymer demixing. Cell spreading, cytoskeleton, focal adhesion and Rac localisation were studied. The results showed that an initial rapid adhesion and cytoskeletal formation on the islands at 4 days of culture gave way to poorly formed contacts and vimentin cytoskeleton at 30 days of culture.

Fibroblast reaction to island topography: changes in cytoskeleton and morphology with time.

In order to develop next-generation tissue engineering materials, the understanding of cell responses to novel material surfaces needs to be better understood. Topography presents powerful cues for cells, and it is becoming clear that cells will react to nanometric, as well as micrometric, scale surface features. Polymer-demixing of polystyrene and polybromostyrene has been found to produce nanoscale islands of reproducible height, and is very cheap and fast compared to techniques such as electron beam lithography. This study observed temporal changes in cell morphology and actin and tubulin cytoskeleton using scanning electron and fluorescence microscopy. The results show large differences in cell response to 95 nm high islands from 5 min to 3 weeks of culture. The results also show a change in cell response from initial fast organisation of cytoskeleton in reaction to the islands, through to lack of cell spreading and low recruitment of cell numbers on the islands.

Polymer-demixed nanotopography: control of fibroblast spreading and proliferation.

Cell response to nanometric scale topography is a growing field. Nanometric topography production has traditionally relied on expensive and time-consuming techniques such as electron beam lithography. This presents disadvantages to the cell biologist in regard to material availability. New research is focusing on less expensive methods of nanotopography production for in vitro cell engineering. One such method is the spontaneous demixing of polymers (in this case polystyrene and polybromostyrene) to produce nanometrically high islands. This article observes fibroblast response to nanometric islands (13, 35, and 95 nm in height) produced by polymer demixing. Changes in cell morphology, cytoskeleton, and proliferation are observed by light, fluorescence, and scanning electron microscopy. Morphological features produced by cells in response to the materials were selected, and cell shape parameters were measured with shape-recognition software. The results showed that island height could either increase or reduce cell spreading and proliferation in relation to control, with 13-nm islands producing cells with the greatest area and 95 nm islands producing cells with the lowest areas. Interaction of filopodia with the islands could been seen to increase as island size was increased.

Interaction of animal cells with ordered nanotopography.

Animal cells live in a complex and diverse environment where they encounter a vast amount of information, a considerable amount of which is in the nanometer range. The surface topography that a cell encounters has a role to play in influencing cell behavior. It has been demonstrated widely that surface shape can directly influence the behavior of cells. In this paper, we discuss the interactions of animal cells with engineered nanotopography, fabricated in quartz and reverse embossed into polycaprolactone, fibroblast cells show reduced adhesion to the ordered nano pits. We show that the area of cells spreading on a structured nanotopography is reduced compared with that on a planar substrate. Furthermore, cytoskeletal organization is disrupted as indicated by a marked decrease in number and size of focal contacts.

Cells react to nanoscale order and symmetry in their surroundings.

Mammalian cells react to microstructured surfaces, but there is little information on the reactions to nanostructured surfaces, and such as have been tested are poorly ordered or random in their structure. We now report that ordered surface arrays (orthogonal or hexagonal) of nanopits in polycaprolactone or polymethylmethacrylate have marked effects in reducing cell adhesion compared with less regular arrays or planar surfaces. The pits had diameters of 35, 75, and 120 nm, respectively, with pitch between the pits of 100, 200, and 300 nm, respectively. The cells appear to be able to distinguish between different symmetries of array. We suggest that interfacial forces may be organized by the nanostructures to affect the cells in the same way as they affect liquid crystal orientations.

Nonadhesive nanotopography: fibroblast response to poly(n-butyl methacrylate)-poly(styrene) demixed surface features.

It is becoming clear that cells do not only respond to micrometric scale topography, but may also respond to topography at the nanometric scale. Nano-fabrication methods such as electron beam lithography are, however, expensive and time consuming. Polymer demixing of poly(styrene) and poly(4-bromostyrene) has been found to produce nano-scale islands of reproducible height, and the islands have been previously shown to effect cell events such as adhesion, spreading, proliferation, and differentiation. This study uses demixed poly(styrene) and poly(n-butyl methacrylate) to produce nano-islands with closer packing and narrower widths compared with those previously studied. Observations have been made of morphological and cytoskeletal changes in human fibroblasts interacting with 10- and 50-nm-high islands. The methods used included scanning electron microscopy, fluorescent microscopy, and optical microscopy. The results indicated that the cells do not respond differently to the 10-nm islands compared with planar samples but, in contrast, the 50-nm islands are nonadhesive.

Cell patterning with a heptagon acoustic tweezer--application in neurite guidance.

Accurate control over positioning of cells is a highly desirable feature in tissue engineering applications since it allows, for example, population of substrates in a controlled fashion, rather than relying on random seeding. Current methods to achieve a differential distribution of cells mostly use passive patterning methods to change chemical, mechanical or topographic properties of surfaces, making areas differentially permissive to the adhesion of cells. However, these methods have no ad hoc control over the actual deposition of cells. Direct patterning methods like bioprinting offer good control over cell position, but require sophisticated instrumentation and are often cost- and time-intensive. Here, we present a novel electronically controlled method of generating dynamic cell patterns by acoustic trapping of cells at a user-determined position, with a heptagonal acoustic tweezer device. We demonstrate the capability of the device to create complex patterns of cells using the device's ability to re-position acoustic traps by using a phase shift in the acoustic wave, and by switching the configuration of active piezoelectric transducers. Furthermore, we show that by arranging Schwann cells from neonatal rats in a linear pattern we are able to create Bands of Büngner-like structures on a non-structured surface and demonstrate that these features are able to guide neurite outgrowth from neonatal rat dorsal root ganglia.

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture.

In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled-down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell-seeding procedures for static cultures might not be applicable, therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro-grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture.

Polyelectrolyte multilayers generated in a microfluidic device with pH gradients direct adhesion and movement of cells.

In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three different pH values of 5, 7 and 9. Water contact angle and quartz microbalance measurements show that resulting multilayers differ in terms of wetting behaviour, layer mass and mechanical properties. The multilayer is then formed within a gradient generation microfluidic (µFL) device. Polyethylene imine or heparin solutions of pH 5 are introduced into one inlet and the same solutions but at pH 9 into another inlet of the µFL device. The pH gradient established during the multilayer formation can be visualized inside the microchamber by pH sensitive fluorophores and confocal laser scanning microscopy. From this setup it is expected that properties of multilayers displayed at distinct pH values can be realised in a gradient manner inside the µFL device. Behaviour of the osteoblast cell line MG-63 seeded and cultured on top of multilayers created inside the µFL device support this hypothesis. It is observed that more cells adhere and spread on multilayers build-up at the basic side of the µFL channel, while those cells on top of multilayers built at pH 5 are fewer and smaller. These results are consistent with the behaviour of MG-63 cells seeded on multilayers formed at discrete pH values. It is particularly interesting to see that cells start to migrate from multilayers built at pH 5 to those built at pH 9 during 6 h of culture. Overall, the presented multilayer formation setup applying pH gradients leads to surfaces that promote migration of cells.

A hierarchical response of cells to perpendicular micro- and nanometric textural cues.

In this paper, we report on the influence of shallow micro- and nanopatterned substrata on the attachment and behavior of a human fibroblast [human telomerase transfected immortalized (hTERT)] cells. We identify a hierarchy of textural guidance cues with respect to cell alignment on these substrates. Cells were seeded and cultured for 48 h on silicon substrates patterned with two linear textures overlaid at 90 degrees, both with 24 microm pitch: a micrograting and a nanopattern of rows of 140- nm-diameter pits arranged in a rectangular array with 300 nm centre-to-centre spacing. The cell response to these textures was shown to be highly dependent on textural feature dimensions. We show that cells align to the stripes of nanopits. Stripes of 30-nm deep nanopits were also shown to elicit a stronger response from cells than 160-nm deep nanopits.

Is surface chemical composition important for orthopaedic implant materials?

Ti-6Al-7Nb (NS) in its 'standard' implant form has been previously shown to be detrimental to fibroblast growth and colonisation on its surface. Specific aspects of the NS topography have been implicated, however, the contribution of its unique surface chemistry to the cell behaviour was unknown. By evaporating either gold or titanium on the surface of standard NS, two different model surface chemistries could be studied with the same characteristic standard NS topography. Two other 'standard' orthopaedic topographies, that of stainless steel (SS) and of 'commercially pure' titanium (TS) were also treated in a similar manner. All materials elicited behaviour similar to their uncoated counterparts. For coated SS and TS, cell proliferation was observed, cells were well spread and displayed mature focal adhesion sites, and associated cytoskeletal components. For coated NS, cell proliferation was compromised, cells remained rounded, filopodia attached and seemed to probe the surface, especially the beta -phase particles, and both the focal adhesion sites and the microtubule network were disrupted by the presence of these particles. These results confirmed, that in the instance of NS, the topography was the primary cause for the observed stunted cell growth. For biomaterials studies, the standardisation of surface chemistry used here is a valuable tool in allowing vastly different materials and surface finishes to be compared solely on the basis of their topography.

3D polymer scaffolds for tissue engineering.

This review discusses some of the most common polymer scaffold fabrication techniques used for tissue engineering applications. Although the field of scaffold fabrication is now well established and advancing at a fast rate, more progress remains to be made, especially in engineering small diameter blood vessels and providing scaffolds that can support deep tissue structures. With this in mind, we introduce two new lithographic methods that we expect to go some way to addressing this problem.

Articular chondrocyte passage number: influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography.

The isolation and culture of articular chondrocytes is a prerequisite of their use in tissue engineering, but prolonged culture and passaging is associated with de-differentiation. In this paper we studied the influence of nanometric and micrometric grooves (85 nm to 8 microm in depth and 2 microm to 20 microm in width) on 1st and 2nd passage ovine chondrocytes since our earlier findings indicate that primary cells are not affected by such features. 1st and 2nd passage chondrocytes cultured on grooved substrata showed a polarisation of cell shape parallel to the groove long axis and F-actin condensations were evident at groove ridge boundaries. An increase in cell migration with increasing groove depth was observed. Both passages of chondrocytes maintained type II collagen expression, but to a lesser degree in 2nd. This study demonstrates that passage number alters the response of chondrocytes to micrometric and nanometric topography, and could be important in ex vivo cartilage engineering.

The response of primary articular chondrocytes to micrometric surface topography and sulphated hyaluronic acid-based matrices.

Understanding the response of chondrocytes to topographical cues and chemical patterns could provide invaluable information to advance the repair of chondral lesions. We studied the response of primary chondrocytes to nano- and micro-grooved surfaces, and sulphated hyaluronic acid (HyalS). Cells were grown on grooves ranging from 80 nm to 9 microm in depth, and from 2 microm to 20 microm in width. Observations showed that the cells did not spread appreciably on any groove size, or alter morphology or F-actin organization, although cells showed accelerated movement on 750 nm deep grooves in comparison to flat surfaces. On chemical patterns, the cells migrated onto, and preferentially attached to, HyalS and showed a greater degree of spreading and F-actin re-arrangement. This study shows that 750 nm deep grooves and sulphated hyaluronic acid elicit responses from primary chondrocytes, and this could have implications for the future direction of cartilage reconstruction and orthopaedic treatments in general.