Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

IL-6 and TGF-ß-Secreting Adoptively-Transferred Murine Mesenchymal Stromal Cells Accelerate Healing of Psoriasis-like Skin Inflammation and Upregulate IL-17A and TGF-ß.

Mesenchymal stromal cells (MSC) show promise as cellular therapeutics. Psoriasis is a chronic inflammatory disease affecting the skin and the joints. Injury, trauma, infection and medications can trigger psoriasis by disrupting epidermal keratinocyte proliferation and differentiation, which activates the innate immune system. Pro-inflammatory cytokine secretion drives a T helper 17 response and an imbalance of regulatory T cells. We hypothesized that MSC adoptive cellular therapy could immunomodulate and suppress the effector T cell hyperactivation that underlies the disease. We used the imiquimod-induced psoriasis-like skin inflammation model to study the therapeutic potential of bone marrow and adipose tissue-derived MSC in vivo. We compared the secretome and the in vivo therapeutic potential of MSC with and without cytokine pre-challenge ("licensing"). The infusion of both unlicensed and licensed MSC accelerated the healing of psoriatic lesions, and reduced epidermal thickness and CD3+ T cell infiltration while promoting the upregulation of IL-17A and TGF-ß. Concomitantly, the expression of keratinocyte differentiation markers in the skin was decreased. However, unlicensed MSC promoted the resolution of skin inflammation more efficiently. We show that MSC adoptive therapy upregulates the transcription and secretion of pro-regenerative and immunomodulatory molecules in the psoriatic lesion. Accelerated healing is associated with the secretion of TGF-ß and IL-6 in the skin and MSC drives the production of IL-17A and restrains T-cell-mediated pathology.

Hamsters Protected from SARS-CoV-2 Delta Variant Challenge after Two Doses of Adjuvanted SARS-CoV-2 Recombinant Spike Protein (S-2P) and One Dose of Beta S-2P.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern negatively impact the effectiveness of vaccines. In this study, we challenge hamsters with the delta variant after 2- or 3-dose inoculations with SARS-CoV-2 vaccines constructed from stabilized prefusion spike proteins (S-2P) of Wuhan (W) and beta (B) variants. Compared to 3 doses of W S-2P, 2 doses of W S-2P followed by a third dose of B S-2P induced the highest neutralizing antibody titer against live SARS-CoV-2 virus and enhanced neutralization of omicron variant pseudovirus. Reduced lung live virus titer and pathology suggested that all vaccination regimens protect hamsters from SARS-CoV-2 delta variant challenge.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

Chemokines and their receptors: predictors of the therapeutic potential of mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSCs) are promising cellular therapeutics for the treatment of inflammatory and degenerative disorders due to their anti-inflammatory, immunomodulatory and regenerative potentials. MSCs can be sourced from a variety of tissues within the body, but bone marrow is the most frequently used starting material for clinical use. The chemokine family contains many regulators of inflammation, cellular function and cellular migration-all critical factors in understanding the potential potency of a novel cellular therapeutic. In this review, we focus on expression of chemokine receptors and chemokine ligands by MSCs isolated from different tissues. We discuss the differential migratory, angiogenetic and immunomodulatory potential to understand the role that tissue source of MSC may play within a clinical context. Furthermore, this is strongly associated with leukocyte recruitment, immunomodulatory potential and T cell inhibition potential and we hypothesize that chemokine profiling can be used to predict the in vivo therapeutic potential of MSCs isolated from new sources and compare them to BM MSCs.

ACL Study Group survey reveals the evolution of anterior cruciate ligament reconstruction graft choice over the past three decades.

PURPOSE: Anterior cruciate ligament reconstruction (ACLR) aims to restore knee function and stability, allowing patients to return to the activities they enjoy and minimize further injury to the meniscus and cartilage and their ultimate progression to osteoarthritis. This study aims to present the evolution of graft choice over the last three decades according to members of the ACL Study Group (SG). METHODS: Prior to the January 2020 ACL SG biannual meeting, a survey was administered consisting of 87 questions and 16 categories, including ACLR graft choice. A similar questionnaire has been administered prior to each meeting and survey results from the past 14 meetings (1992 through 2020, excluding 1994) are included in this work. Survey responses are reported as frequencies in percentages to quantify changes in practice over the surgery period. RESULTS: In 1992, the most frequent graft choice for primary ACLR was bone-patellar tendon-bone (BTB) autograft, at nearly 90%. Hamstring tendon (HT) autografts have increased in popularity, currently over 50%, followed by just under 40% BTB autograft. Recently, quadriceps tendon (QT) autograft has increased in popularity since 2014. CONCLUSION: Autograft (HT, BTB, QT) is an overwhelming favorite for primary ACLR over allograft. The preference for HT autograft increased over the study period relative to BTB autograft, with QT autograft gaining in popularity in recent years. Graft selection should be individualized for each patient and understanding the global trends in graft choice can help orthopaedic surgeons discuss graft options with their patients and determine the appropriate graft for each case. LEVEL OF EVIDENCE: Level V, Expert Opinion.

The use of mesenchymal stromal cells in the treatment of coronavirus disease 2019.

More than seven months into the coronavirus disease -19 (COVID-19) pandemic, infection from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to over 21.2 million cases and resulted in over 760,000 deaths worldwide so far. As a result, COVID-19 has changed all our lives as we battle to curtail the spread of the infection in the absence of specific therapies against coronaviruses and in anticipation of a proven safe and efficacious vaccine. Common with previous outbreaks of coronavirus infections, SARS and Middle East respiratory syndrome, COVID-19 can lead to acute respiratory distress syndrome (ARDS) that arises due to an imbalanced immune response. While several repurposed antiviral and host-response drugs are under examination as potential treatments, other novel therapeutics are also being explored to alleviate the effects on critically ill patients. The use of mesenchymal stromal cells (MSCs) for COVID-19 has become an attractive avenue down which almost 70 different clinical trial teams have ventured. Successfully trialled for the treatment of other conditions such as multiple sclerosis, osteoarthritis and graft versus host disease, MSCs possess both regenerative and immunomodulatory properties, the latter of which can be harnessed to reduce the severity and longevity of ARDS in patients under intensive care due to SARS-CoV-2 infection.

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). METHODS: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. RESULTS: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. CONCLUSIONS: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.

A CpG-Ficoll Nanoparticle Adjuvant for Anthrax Protective Antigen Enhances Immunogenicity and Provides Single-Immunization Protection against Inhaled Anthrax in Monkeys.

Nanoparticulate delivery systems for vaccine adjuvants, designed to enhance targeting of secondary lymphoid organs and activation of APCs, have shown substantial promise for enhanced immunopotentiation. We investigated the adjuvant activity of synthetic oligonucleotides containing CpG-rich motifs linked to the sucrose polymer Ficoll, forming soluble 50-nm particles (DV230-Ficoll), each containing >100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using recombinant protective Ag (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing Abs in cynomolgus monkeys at 2 wk compared with animals immunized with equivalent amounts of monomeric DV230. Monkeys immunized either once or twice with rPA plus DV230-Ficoll were completely protected from challenge with 200 LD50 aerosolized anthrax spores. In mice, DV230-Ficoll was more potent than DV230 for the induction of innate immune responses at the injection site and draining lymph nodes. DV230-Ficoll was preferentially colocalized with rPA in key APC populations and induced greater maturation marker expression (CD69 and CD86) on these cells and stronger germinal center B and T cell responses, relative to DV230. DV230-Ficoll was also preferentially retained at the injection site and draining lymph nodes and produced fewer systemic inflammatory responses. These findings support the development of DV230-Ficoll as an adjuvant platform, particularly for vaccines such as for anthrax, for which rapid induction of protective immunity and memory with a single injection is very important.

Development, functional characterization and validation of methodology for GMP-compliant manufacture of phagocytic macrophages: A novel cellular therapeutic for liver cirrhosis.

BACKGROUND AIMS: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial). METHODS: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14+ cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product. RESULTS: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest. CONCLUSIONS: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen.

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

Chemokines as novel and versatile reagents for flow cytometry and cell sorting.

Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications.

Mesenchymal stromal cells as multifunctional cellular therapeutics - a potential role for extracellular vesicles.

Mesenchymal stromal cells (MSCs), multipotent cells present in tissues throughout the body, can reconstitute adipogenic, osteogenic and chondrogenic tissues, but are also of great interest as mediators of immune modulation and suppression. MSCs are able to improve transplant engraftment, treat graft versus host disease and suppress T cell responses and therefore have great potential as therapeutic agents. Their immune modulatory capacity is mediated through both cell-to-cell contact and cytokine secretion, but it is becoming clear that extracellular vesicles (EV) produced by MSC also possess immunomodulatory properties. These vesicles are easy to prepare and store, do not carry nuclear material and cannot form tumours, and therefore also represent a highly desirable therapeutic agent. This review outlines the formation and characterisation of extracellular vesicles, the reported function of MSC-EVs in vitro and in vivo, and addresses some of the emerging issues with nomenclature, EV therapeutic dose and tissue source. The development of GMP-grade production protocols and effective characterisation of MSC extracellular vesicles is essential to their successful use as immune modulating therapeutic agents, and this review outlines the current status of the research in this area.

Islet transplantation from a nationally funded UK centre reaches socially deprived groups and improves metabolic outcomes.

AIMS/HYPOTHESIS: Type 1 diabetes complicated by hypoglycaemia is prevalent in socioeconomically deprived populations. Islet transplantation is of proven efficacy in type 1 diabetes complicated by hypoglycaemia, but it is not known if nationally funded programmes reach the socioeconomically deprived. Our aim was to determine: (1) socioeconomic indices in participants referred to our nationally funded programme; and (2) if metabolic outcomes in our transplant recipients were improved. METHODS: Participants referred (n = 106) and receiving transplants (n = 18; 32 infusions) were examined with respect to socioeconomic status (deprivation category score) and their ability to work and drive. In participants followed for ≥12 months after transplantation, metabolic and anthropometric measurements (n = 14) were recorded pre- and post-transplant (assessed ~1, ~3, ~6 and ~12 months with mixed-meal tolerance tests and 6 day continuous glucose monitoring assessments). Donor data was also examined. RESULTS: There was a greater prevalence of socioeconomic deprivation in referred and transplant recipients than the general population (p < 0.05). Of the transplant recipients, 73% were socioeconomically deprived, 88% did not hold a driver's license and 94% had reduced ability to work (all p < 0.01 vs referred participants). Donors were predominantly obese and included circulatory death donors. At 12 months, 93% of participants who had received transplants had graft function, diminished frequency of hypoglycaemia (10 [4-11] vs 0 [0-2] hypoglycaemic episodes/week), improved awareness of hypoglycaemia (Gold score 7 [5-7] vs 1 [1-2]) and glycaemic control (HbA1c: 7.9% [7.2-8.5%]; 63 [55-69] mmol/mol vs 7.2% [6.8-7.5%]; 55 [51-58] mmol/mol), diminished glycaemic lability and decreased central adiposity (all p < 0.05). CONCLUSIONS/INTERPRETATION: A nationally funded islet transplant programme reaches the socioeconomically deprived and outcomes are significantly improved in this group.

Phenotypic and functional characterization of macrophages with therapeutic potential generated from human cirrhotic monocytes in a cohort study.

BACKGROUND AIMS: Macrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis. METHODS: Infusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-ß, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases [MMP]-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice-compatible technique. RESULTS: There was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 10(8) and 2.5 ± 0.56 × 10(8), respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 10(8) ± 0.38 × 10(8), with more than 90% viability and 0.65 × 10(8) ± 0.16 × 10(8), respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin [IL]-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences. CONCLUSIONS: Macrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy.

A limited CpG-containing oligodeoxynucleotide therapy regimen induces sustained suppression of allergic airway inflammation in mice.

BACKGROUND: CpG-containing oligodeoxynucleotides (CpG-ODNs) are potent inhibitors of T helper 2 mediated allergic airway disease in sensitised mice challenged with allergen. A single treatment has transient effects but a limited series of treatments has potential to achieve clinically meaningful sustained inhibition of allergic airway disease. OBJECTIVE: To optimise the treatment regimen for sustained efficacy and to determine the mechanisms of action in mice of an inhaled form of CpG-ODN being developed for human asthma treatment. METHODS: We set up a chronic allergic-asthma model using ragweed-sensitised mice exposed weekly to intranasal ragweed. Using this model, the effects of a limited series of weekly intranasal 1018 ISS (CpG-ODN; B-class) treatments were evaluated during treatment and for several weeks after treatments had stopped but weekly allergen exposures continued. Treatment efficacy was evaluated by measuring effects on lung T helper 2 cytokines and eosinophilia, and lung dendritic cell function and T-cell responses. RESULTS: Twelve intranasal 1018 ISS treatments induced significant suppression of bronchoalveolar lavage eosinophilia and interleukin 4, 5 and 13 levels. This suppression of allergic T helper 2 parameters was maintained through 13 weekly ragweed exposures administered after treatment cessation. Subsequent experiments demonstrated that at least five treatments were required for lasting suppression. Although CpG-ODN induced moderate T helper 1 responses, suppression of allergic airway disease did not require interferon γ but was associated with induction of a regulatory T-cell response. CONCLUSIONS: A short series of CpG-ODN treatments results in sustained suppression of allergic lung inflammation induced by a clinically relevant allergen.

Human leucocytes processed by fast-rate inertial microfluidics retain conventional functional characteristics.

The manufacturing of clinical cellular therapies is a complex process frequently requiring manipulation of cells, exchange of buffers and volume reduction. Current manufacturing processes rely on either low throughput open centrifugation-based devices, or expensive closed-process alternatives. Inertial focusing (IF) microfluidic devices offer the potential for high-throughput, inexpensive equipment which can be integrated into a closed system, but to date no IF devices have been approved for use in cell therapy manufacturing, and there is limited evidence for the effects that IF processing has on human cells. The IF device described in this study was designed to simultaneously separate leucocytes, perform buffer exchange and provide a volume reduction to the cell suspension, using high flow rates with high Reynolds numbers. The performance and effects of the IF device were characterized using peripheral blood mononuclear cells and isolated monocytes. Post-processing cell effects were investigated using multi-parameter flow cytometry to track cell viability, functional changes and fate. The IF device was highly efficient at separating CD14+ monocytes (approx. 97% to one outlet, approx. 60% buffer exchange, 15 ml min-1) and leucocyte processing was well tolerated with no significant differences in downstream viability, immunophenotype or metabolic activity when compared with leucocytes processed with conventional processing techniques. This detailed approach provides robust evidence that IF devices could offer significant benefits to clinical cell therapy manufacture.

Meniscal and Articular Cartilage Predictors of Outcome After Revision ACL Reconstruction: A 6-Year Follow-up Cohort Study.

BACKGROUND: Meniscal and chondral damage is common in the patient undergoing revision anterior cruciate ligament (ACL) reconstruction. PURPOSE: To determine if meniscal and/or articular cartilage pathology at the time of revision ACL surgery significantly influences a patient's outcome at 6-year follow-up. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Patients undergoing revision ACL reconstruction were prospectively enrolled between 2006 and 2011. Data collection included baseline demographics, surgical technique, pathology, treatment, and scores from 4 validated patient-reported outcome instruments: International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Marx Activity Rating Scale. Patients were followed up at 6 years and asked to complete the identical set of outcome instruments. Regression analysis assessed the meniscal and articular cartilage pathology risk factors for clinical outcomes 6 years after revision ACL reconstruction. RESULTS: An overall 1234 patients were enrolled (716 males, 58%; median age, 26 years). Surgeons reported the pathology at the time of revision surgery in the medial meniscus (45%), lateral meniscus (36%), medial femoral condyle (43%), lateral femoral condyle (29%), medial tibial plateau (11%), lateral tibial plateau (17%), patella (30%), and trochlea (21%). Six-year follow-up was obtained on 79% of the sample (980/1234). Meniscal pathology and articular cartilage pathology (medial femoral condyle, lateral femoral condyle, lateral tibial plateau, trochlea, and patella) were significant drivers of poorer patient-reported outcomes at 6 years (IKDC, KOOS, WOMAC, and Marx). The most consistent factors driving outcomes were having a medial meniscal excision (either before or at the time of revision surgery) and patellofemoral articular cartilage pathology. Six-year Marx activity levels were negatively affected by having either a repair/excision of the medial meniscus (odds ratio range, 1.45-1.72; P≤ .04) or grade 3-4 patellar chondrosis (odds ratio, 1.72; P = .04). Meniscal pathology occurring before the index revision surgery negatively affected scores on all KOOS subscales except for sports/recreation (P < .05). Articular cartilage pathology significantly impaired all KOOS subscale scores (P < .05). Lower baseline outcome scores, higher body mass index, being a smoker, and incurring subsequent surgery all significantly increased the odds of reporting poorer clinical outcomes at 6 years. CONCLUSION: Meniscal and chondral pathology at the time of revision ACL reconstruction has continued significant detrimental effects on patient-reported outcomes at 6 years after revision surgery.