Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible electroporation for ...cryopreservation of two types of human mesenchymal stromal cells was investigated: adipose-derived stromal cells, and umbilical-cord-derived stromal cells. Comparable results to standard dimethyl sulfoxide cryopreservation protocols were achieved, even without extensive electroporation parameters and protocol optimization. The presence of high extracellular trehalose resulted in comparable cell viabilities without and with electroporation. According to the determination of trehalose concentrations, 250 mM extracellular trehalose resulting in, 20 mM to 50 mM intracellular trehalose were sufficient for successful cryopreservation of cells. With electroporation, higher (i.e. 50 mM to 90 mM) intracellular trehalose was achieved after cryopreservation, although cell survival was not improved significantly. To evaluate the impact of electroporation and cryopreservation on cells, stress and immune-activation-related gene expression were analyzed. Electroporation and/or cryopreservation resulted in increased
SOD2
and
HSPA1A
expression. Despite the increased stress response, the high up-regulation by mesenchymal stromal cells of immunomodulatory genes in the inflammatory environment was not affected. Highest expression was seen for the
IDO1
and
TSG6
genes. In conclusion, cryopreservation of mesenchymal stromal cells in trehalose results in comparable characteristics to their cryopreservation using dimethyl sulfoxide.
Purpose To evaluate the hypothetical toxic effect of local anesthetics on the articular cartilage using patient data from autologous chondrocyte cultivation with different anesthesia types used for ...arthroscopic cartilage biopsy specimen procurement. Methods A retrospective analysis of patient data from the national autologous chondrocyte implantation registry and the corresponding hospital records was approved by the National Medical Ethics Committee. Articular cartilage biopsy specimens from the knees of 49 consecutive patients assigned for autologous chondrocyte implantation (aged 14 to 44 years) were procured from the non–weight-bearing articular surface during arthroscopy under general anesthesia (12 patients), spinal anesthesia (18 patients), or local anesthesia (intra-articular injection of 15 to 20 mL of 2% lidocaine hydrochloride) (19 patients). All the biopsy specimens were further manipulated following the same chondrocyte cultivation protocol. General patient data and surgery-related parameters, together with chondrocyte viability, population doublings, and chondrocyte morphology in biopsy specimens and primary cell cultures, were analyzed and compared across different types of anesthesia. Results Patients in the general, spinal, and local anesthesia groups showed no statistical differences in age (27 years, 29 years, and 32 years, respectively), duration of surgery (36 minutes, 37 minutes, and 39 minutes, respectively), weight of biopsy specimens (110 mg, 178 mg, and 130 mg, respectively), cell viability in cartilage biopsy specimens (67%, 69%, and 78%, respectively) or primary cultures (95%, 95%, and 95%, respectively), and population doublings (5.2, 5.2, and 5.2, respectively). Similar chondrocyte morphology in primary cell cultures was observed among the 3 groups. Conclusions This retrospective study showed that a single intra-articular injection of lidocaine hydrochloride used for knee arthroscopy did not influence the viability, morphology, and cultivation potential of chondrocytes in articular cartilage biopsy specimens assigned for autologous chondrocyte implantation. Level of Evidence Level IV, retrospective comparative study.
This work reports about the conjugation of glycine C-terminal ethyl and methyl ester peptides and L-tryptophan methyl ester with sodium hyaluronate in aqueous solutions using the peptide coupling ...agent DMTMM (or short DMT, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride). Detailed infrared (IR) absorbance and 1H and 13C (2D) NMR studies (heteronuclear multi-bond correlation spectroscopy, HMBC) confirmed covalent and regioselective amide bonds with the D-glucuronate, but also proves the presence of DMT traces in all conjugates. The ethyl ester`s methyl protons on the peptides` C-terminal could be used to quantify the degree of substitution of the peptide on the hyaluronate scaffold by NMR. The ester group also proved stable during conjugation and work-up, and could in some cases be selectively cleaved in water whilst leaving the amide bond intact as shown by potentiometric charge titration, NMR and IR. The conjugates did not influence the capability of human umbilical vein endothelial cells (HUVECs) to reduce MTS (5-3-(carboxymethoxy)phenyl-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) to a formazan dye, which points towards a low cytotoxicity for the obtained products. The conjugation method and products could be tested for tissue engineering gels or drug delivery purposes with alternative, biologically active peptides.
New cryopreservation approaches for medically applicable cells are of great importance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide (DMSO), which is toxic to cells and ...causes undesirable side effects in patients, such as cardiac arrhythmias, neurological events, and others. Trehalose, a nontoxic disaccharide, has been already studied as a cryoprotectant. However, an efficient approach for loading this impermeable sugar into mammalian cells is missing. In our study, we assessed the efficiency of combining reversible electroporation and trehalose for cryopreservation of human adipose-derived stem cells. First, we determined reversible electroporation threshold by loading of propidium iodide into cells. The highest permeabilization while maintaining high cell viability was reached at 1.5 kV/cm, at 8 pulses, 100 µs, and 1 Hz. Second, cells were incubated in 250 or 400 mM trehalose and electroporated before cryopreservation. After thawing, 83.8 ± 1.8 % (mean ± SE) cell recovery was obtained at 250 mM trehalose. By using a standard freezing protocol (10 % DMSO in 90 % fetal bovine serum), cell survival after thawing was about 91.5 ± 1.6 %. We also evaluated possible effects of electroporation on cells’ functionality before and after thawing. Successful cell growth and efficient adipogenic and osteogenic differentiation were achieved. In conclusion, electroporation seems to be an efficient method for loading nonpermeable trehalose into human adipose-derived stem cells, allowing long-term cryopreservation in DMSO-free and xeno-free conditions.
Purpose
To document clinical, radiologic, and cellular data of a prospective patient series treated by a tri-layer collagen-hydroxyapatite biomimetic osteochondral scaffold (CHAS) intra-operatively ...seeded with cultivated autologous chondrocytes (AC) or with filtered bone marrow stem/stromal cells (fBMSC) to address chronic osteochondral knee lesions.
Methods
Thirty-six consecutive patients (15 to 59 years) with chronic osteochondral lesions (1.8–10 cm
2
) in the condylar or patellofemoral knee surfaces were enrolled. Lesions were covered with CHAS fixed with a fibrin glue. The superficial layer of CHAS was intra-operatively injected with active cells: in initial five patients, ACs were put directly onto dry CHAS (dry-AC); next, eight AC patients had CHAS moistened with cell culture media (media-AC), while the tourniquet was released allowing blood soaking of CHAS in the rest (14 blood-AC, 9 blood-fBMSC). Seventeen (50%) patients required different concomitant procedures. All patients were followed for serious adverse events (SAE) or graft failures; clinical, radiographic, and MRI evaluation was conducted. Cellular data on the injected cells were assessed.
Results
At a follow-up of 39 months (16–81), 17 patients required an additional surgical intervention: seven graft-related SAE (early post-operative synovitis and/or arthrofibrosis) were registered (3 dry-AC, 3 media-AC, 1 blood-fBMSC). There were two graft failures (1 dry-AC, 1 blood-fBMSC) for secondary reasons. All clinical scores significantly improved from pre- to post-operative values: IKCD subjective 44 to 65; IKDC examination (9/17/5/5) to (20/10/5/1); KOOS (P61/S59/ADL67/Sp32/QoL31) to (P79/S75/ADL84/Sp55/QoL51); Tegner activity scale 3.3 to 4.4. There was evidence of radiographic osteoarthritis progression—Kellgren-Lawrence 1.0 to 1.5. MOCART scores at the final follow-up averaged 71 (10 to 95). Graft-type analysis demonstrated an increased rate of graft-related SAE in dry-AC and media-AC, but their final outcomes were equivalent. Cellular data of AC at the implantation were as follows: cells in suspension 9.2 × 10
6
, viability 95%. In blood-fBMSC group, a cell suspension with 87% viability was injected, which contained 1156 CFU-Fs.
Conclusion
CHAS with intra-operative seeding of active cells, either AC or fBMSC, led to an overall successful outcome for the treatment of chronic osteochondral lesions in the knee. Blood soaking of CHAS in situ before cell seeding significantly decreased early post-operative adverse events, such as synovitis and arthrofibrosis.
The musculoskeletal system includes tissues that have remarkable regenerative capabilities. Bone and muscle sustain micro-damage throughout the lifetime, yet they continue to provide the body with ...the support that is needed for everyday activities. Our current understanding is that the regenerative capacity of the musculoskeletal system can be attributed to the mesenchymal stem/ stromal cells (MSCs) that reside within its different anatomical compartments. These MSCs can replenish various tissues with progenitor cells to form functional cells, such as osteoblasts, chondrocytes, myocytes, and others. However, with aging and in certain disorders of the musculoskeletal system such as osteoarthritis or osteoporosis, this regenerative capacity of MSCs appears to be lost or diverted for the production of other non-functional cell types, such as adipocytes and fibroblasts. In this review, we shed light on the tissue sources and subpopulations of MSCs in the musculoskeletal system that have been identified in animal models, discuss the mechanisms of their anti-inflammatory action as a prerequisite for their tissue regeneration and their current applications in regenerative medicine. While providing up-to-date evidence of the role of MSCs in different musculoskeletal pathologies, in particular in osteoporosis and osteoarthritis, we share some thoughts on their potential as diagnostic markers in musculoskeletal health and disease.
Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard ...freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum.
In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).
Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.
Actinia fragacea is commonly called the “strawberry” anemone because of the distinctive yellow or green spots displayed on its red column. Its venom contains several haemolytic proteins with a ...molecular mass of ∼20
kDa that can be separated by ion-exchange column chromatography. One of them was purified to homogeneity and was named fragaceatoxin C (FraC). Its 15 N-terminal residues were identified by Edman degradation and served to obtain its complete DNA coding sequence by RT-PCR. The coding region of FraC was amplified and cloned in the expression vector pBAT-4. Purified recombinant FraC consists of 179 amino acids and multiple sequence alignment with other actinoporins clearly indicates that FraC belongs to this protein family. The secondary structure, thermal stability and lytic activity of native and recombinant FraC were practically identical and exhibit the same basic features already described for equinatoxin-II and sticholysin-II.
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