Intervertebral disc decellularisation: progress and challenges.

Intervertebral disc (IVD) degeneration and the consequent low-back pain (LBP) affect over 80 % of people in western societies, constituting a tremendous socio-economic burden worldwide and largely impairing patients' life quality. Extracellular matrix (ECM)-based scaffolds, derived from decellularised tissues, are being increasingly explored in regenerative medicine for tissue repair. Decellularisation plays an essential role for host cells and antigen removal, while maintaining native microenvironmental signals, including ECM structure, composition and mechanical properties, which are essential for driving tissue regeneration. With the lack of clinical solutions for IVD repair/regeneration, implantation of decellularised IVD tissues has been explored to halt and/or revert the degenerative cascade and the associated LBP symptoms. Over the last few years, several researchers have focused on the optimisation of IVD decellularisation methods, combining physical, chemical and enzymatic treatments, in order to successfully develop a cell-free matrix. Recellularisation of IVD-based scaffolds with different cell types has been attempted and numerous methods have been explored to address proper IVD regeneration. Herein, the advances in IVD decellularisation methods, sterilisation procedures, repopulation and biocompatibility tests are reviewed. Additionally, the importance of the donor profile for therapeutic success is also addressed. Finally, the perspectives and major hurdles for clinical use of the decellularised ECM-based biomaterials for IVD are discussed. The studies reviewed support the notion that tissue-engineering-based strategies resorting to decellularised IVD may represent a major advancement in the treatment of disc degeneration and consequent LBP.

Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use.

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.

Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy.

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br), and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations), to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD, n=30) nucleated cells and was enriched in cells expressing CD34, CD117, CD105, CD127, CD133 and CD166, and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells, and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes, osteoblasts and chondrocytes. DISCUSSION: Hematopoietic, endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications.

A whole-blood assay for qualitative and semiquantitative measurements of CD69 surface expression on CD4 and CD8 T lymphocytes using flow cytometry.

A whole-blood flow cytometry-based assay was utilized to assess CD4 and CD8 T-lymphocyte activation in response to phytohemagglutinin (PHA) stimulation. T-lymphocyte activation was assessed by qualitative (percent CD69) and semiquantitative (anti-CD69 antibody binding capacity) measurements of CD69 surface expression. Whole-blood samples from 21 healthy and 21 human immunodeficiency virus (HIV)-infected (<500 absolute CD4 counts per mm3) individuals were stimulated with 20 microg of PHA per ml for 18 to 24 h. The proportions of activated CD4 and CD8 T lymphocytes expressing CD69 (percent CD69) and the levels of CD69 expression on each T-lymphocyte subset (anti-CD69 antibody binding capacity) were measured. By using this assay system, T-lymphocyte activation was impaired in both CD4 and CD8 T-lymphocyte subsets of HIV-infected individuals. The proportions of CD69-positive CD4 and CD8 T lymphocytes were 43 and 27% lower, respectively, in samples from HIV-infected individuals compared to samples from healthy individuals. Similarly, the levels of CD69 expression on each activated CD4 and CD8 T-lymphocyte subset were 48 and 51% lower, respectively. These results suggest that both qualitative and semiquantitative measurements of CD69 surface expression by flow cytometry can be used to assess T-lymphocyte activation.

Macrophage apoptosis in the absence of active interleukin-1 beta-converting enzyme.

The interleukin-1 beta converting enzyme (ICE) is the cysteine proteinase responsible for cleaving the 31-kDa interleukin-1 beta (IL-1 beta) precursor to its active 17-kDa form. In lipopolysaccharide-stimulated cultured macrophages, induction of apoptosis but not necrosis effectively induces conversion of the IL-1 beta precursor to its mature form and results in the concomitant release of the mature cytokine from the cell. To determine whether ICE activity is required for macrophage apoptosis, we have exposed macrophages either to 5 mM ATP or to alloreactive cytolytic T lymphocytes (CTL) in the absence and presence of the ICE inhibitor peptide YVAD-chloromethylketone (YVAD-emk). Activated cells treated with YVAD-emk and ATP or CTL showed no mature IL-1 beta in either the cell lysates or the culture supernatants, indicating effective inhibition of ICE activity; however, the YVAD-treated macrophages showed no detectable change in 51Cr release or nuclear fragmentation, indicating failure to inhibit apoptotic cell death. Thus, in these cells, YVAD-emk uncouples IL-1 beta processing and apoptosis.