Supercooled preservation of cultured primary rat hepatocyte monolayers.

Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.

Eficacia del trasplante de células madre mesenquimales de lipoaspirado en el tratamiento de lesiones crónicas del manguito rotador. Modelo experimental en ratas / Efficacy of transplantation of lipoaspired mesenchymal stem cells in the treatment of chronic rotator cuff tears. Experimental model in rats

Antecedentes y objetivo: Las roturas del manguito rotador afectan aproximadamente al 30% de la población mayor de 60 años. El tratamiento quirúrgico por vía artroscópica de estas lesiones es el tratamiento de elección, sin embargo, a pesar de las mejoras técnicas de reparación el índice de rerroturas oscila entre el 11 y el 94%. Por ello, los investigadores buscan mejorar el proceso de curación biológica mediante el uso de diferentes alternativas como las células madre mesenquimales (MSC). Nuestro objetivo es evaluar la eficacia de un medicamento de terapia celular elaborado a partir de células madre alogénicas derivadas del tejido adiposo en un modelo de lesión crónica del manguito rotador en ratas. Material y método: Se creó la lesión del supraespinoso en 48 ratas para su posterior sutura a las 4 semanas. A 24 animales se les añadió las MSC en suspensión tras la sutura, y a 24 animales HypoThermosol-FRS® (HTS) como grupo control. En ambos grupos se analizó la histología (escala Åström y Rausing) y la carga máxima, desplazamiento y constante elástica del tendón supraespinoso a los 4 meses de la reparación. Resultados: No se encontraron diferencias estadísticamente significativas en la puntuación histológica comparando los tendones tratados con MSC con respecto a los tendones tratados con HTS (p=0,811) ni tampoco en los resultados de carga máxima (p=0,770), desplazamiento (p=0,852) ni constante elástica (p=0,669) del tendón en ambos grupos. Conclusiones: La adición en suspensión de células derivadas de tejido adiposo a la reparación de una lesión crónica de manguito no mejora las características histológicas ni biomecánicas del tendón suturado.(AU)

Background and aim: Rotator cuff tears emerge in approximately 30% of the population over 60 years of age. Arthroscopic surgical treatment of these lesions is the treatment of choice, however, despite the improved repair techniques, the rate of re-tears ranges between 11 and 94%. Therefore, researchers seek to improve the biological healing process through the use of different alternatives such as mesenchymal stem cells (MSCs). Our objective is to evaluate the efficacy of a Cellular Therapy Drug made from allogeneic stem cells derived from adipose tissue in a rat model of chronic rotator cuff injury. Material and methods: The supraspinatus lesion was created in 48 rats for subsequent suturing at 4 weeks. MSCs in suspension were added to 24 animals after suturing, and HypoThermosol-FRS® (HTS) to 24 animals as a control group. Histology (Åström and Rausing scale) and the maximum load, displacement and elastic constant of the supraspinatus tendon were analyzed in both groups 4 months after the repair. Results: No statistically significant differences were found in the histological score comparing the tendons treated with MSCs with respect to the tendons treated with HTS (P=.811) nor in the results of maximum load (P=.770), displacement (P=.852) or elastic constant (P=.669) of the tendon in both groups. Conclusions: The addition of adipose-derived cells in suspension to the repair of a chronic cuff injury does not improve the histology or biomechanics of the sutured tendon.(AU)

[Translated article] Efficacy of transplantation of lipoaspired mesenchymal stem cells in the treatment of chronic rotator cuff tears. Experimental model in rats / Eficacia del trasplante de células madre mesenquimales de lipoaspirado en el tratamiento de lesiones crónicas del manguito rotador. Modelo experimental en ratas

Antecedentes y objetivo: Las roturas del manguito rotador afectan aproximadamente al 30% de la población mayor de 60 años. El tratamiento quirúrgico por vía artroscópica de estas lesiones es el tratamiento de elección, sin embargo, a pesar de las mejoras técnicas de reparación el índice de rerroturas oscila entre el 11 y el 94%. Por ello, los investigadores buscan mejorar el proceso de curación biológica mediante el uso de diferentes alternativas como las células madre mesenquimales (MSC). Nuestro objetivo es evaluar la eficacia de un medicamento de terapia celular elaborado a partir de células madre alogénicas derivadas del tejido adiposo en un modelo de lesión crónica del manguito rotador en ratas. Material y método: Se creó la lesión del supraespinoso en 48 ratas para su posterior sutura a las 4 semanas. A 24 animales se les añadió las MSC en suspensión tras la sutura, y a 24 animales HypoThermosol-FRS® (HTS) como grupo control. En ambos grupos se analizó la histología (escala Åström y Rausing) y la carga máxima, desplazamiento y constante elástica del tendón supraespinoso a los 4 meses de la reparación. Resultados: No se encontraron diferencias estadísticamente significativas en la puntuación histológica comparando los tendones tratados con MSC con respecto a los tendones tratados con HTS (p=0,811) ni tampoco en los resultados de carga máxima (p=0,770), desplazamiento (p=0,852) ni constante elástica (p=0,669) del tendón en ambos grupos. Conclusiones: La adición en suspensión de células derivadas de tejido adiposo a la reparación de una lesión crónica de manguito no mejora las características histológicas ni biomecánicas del tendón suturado.(AU)

Background and aim: Rotator cuff tears emerge in approximately 30% of the population over 60 years of age. Arthroscopic surgical treatment of these lesions is the treatment of choice, however, despite the improved repair techniques, the rate of re-tears ranges between 11 and 94%. Therefore, researchers seek to improve the biological healing process through the use of different alternatives such as mesenchymal stem cells (MSCs). Our objective is to evaluate the efficacy of a Cellular Therapy Drug made from allogeneic stem cells derived from adipose tissue in a rat model of chronic rotator cuff injury. Material and methods: The supraspinatus lesion was created in 48 rats for subsequent suturing at 4 weeks. MSCs in suspension were added to 24 animals after suturing, and HypoThermosol-FRS® (HTS) to 24 animals as a control group. Histology (Åström and Rausing scale) and the maximum load, displacement and elastic constant of the supraspinatus tendon were analyzed in both groups 4 months after the repair. Results: No statistically significant differences were found in the histological score comparing the tendons treated with MSCs with respect to the tendons treated with HTS (P=.811) nor in the results of maximum load (P=.770), displacement (P=.852) or elastic constant (P=.669) of the tendon in both groups. Conclusions: The addition of adipose-derived cells in suspension to the repair of a chronic cuff injury does not improve the histology or biomechanics of the sutured tendon.(AU)

[Translated article] Efficacy of transplantation of lipoaspired mesenchymal stem cells in the treatment of chronic rotator cuff tears. Experimental model in rats.

BACKGROUND AND AIM: Rotator cuff tears emerge in approximately 30% of the population over 60 years of age. Arthroscopic surgical treatment of these lesions is the treatment of choice, however, despite the improved repair techniques, the rate of re-tears ranges between 11 and 94%. Therefore, researchers seek to improve the biological healing process through the use of different alternatives such as mesenchymal stem cells (MSCs). Our objective is to evaluate the efficacy of a cellular therapy drug made from allogeneic stem cells derived from adipose tissue in a rat model of chronic rotator cuff injury. MATERIAL AND METHODS: The supraspinatus lesion was created in 48 rats for subsequent suturing at 4 weeks. MSCs in suspension were added to 24 animals after suturing, and HypoThermosol-FRS® (HTS) to 24 animals as a control group. Histology (Åström and Rausing scale) and the maximum load, displacement and elastic constant of the supraspinatus tendon were analysed in both groups 4 months after the repair. RESULTS: No statistically significant differences were found in the histological score comparing the tendons treated with MSCs with respect to the tendons treated with HTS (P=0.811) nor in the results of maximum load (P=0.770), displacement (P=0.852) or elastic constant (P=0.669) of the tendon in both groups. CONCLUSIONS: The addition of adipose-derived cells in suspension to the repair of a chronic cuff injury does not improve the histology or biomechanics of the sutured tendon.

Efficacy of transplantation of lipoaspired mesenchymal stem cells in the treatment of chronic rotator cuff tears. Experimental model in rats. / Eficacia del trasplante de células madre mesenquimales de lipoaspirado en el tratamiento de lesiones crónicas del manguito rotador. Modelo experimental en ratas.

BACKGROUND AND AIM: Rotator cuff tears emerge in approximately 30% of the population over 60 years of age. Arthroscopic surgical treatment of these lesions is the treatment of choice, however, despite the improved repair techniques, the rate of re-tears ranges between 11 and 94%. Therefore, researchers seek to improve the biological healing process through the use of different alternatives such as mesenchymal stem cells (MSCs). Our objective is to evaluate the efficacy of a Cellular Therapy Drug made from allogeneic stem cells derived from adipose tissue in a rat model of chronic rotator cuff injury. MATERIAL AND METHODS: The supraspinatus lesion was created in 48 rats for subsequent suturing at 4 weeks. MSCs in suspension were added to 24 animals after suturing, and HypoThermosol-FRS® (HTS) to 24 animals as a control group. Histology (Åström and Rausing scale) and the maximum load, displacement and elastic constant of the supraspinatus tendon were analyzed in both groups 4 months after the repair. RESULTS: No statistically significant differences were found in the histological score comparing the tendons treated with MSCs with respect to the tendons treated with HTS (P=.811) nor in the results of maximum load (P=.770), displacement (P=.852) or elastic constant (P=.669) of the tendon in both groups. CONCLUSIONS: The addition of adipose-derived cells in suspension to the repair of a chronic cuff injury does not improve the histology or biomechanics of the sutured tendon.

Development of supercooling preservation method of adherent cultured human cells.

Cryopreservation of mammalian cells is an important technology; however, freezing damage due to osmotic pressure differences and ice crystal formation is inevitable. In addition, cryopreserved cells cannot be used immediately after thawing in many cases. Therefore, in this study, we developed a method for supercooling and preserving adherent cells using a precision temperature-controlled CO2 incubator. The effects of the cooling rate from 37 to -4°C, the warming rate from -4 to 37°C and a preservation solution on cell viability after storage were examined. Human hepatocarcinoma-derived cell line HepG2 cells, preserved with HypoThermosol FRS at -4°C with a cooling rate of -0.028°C/min (24 h from 37°C to -4°C) and warming to 37°C at a rate of +1.0°C/min (40 min from -4 to 37°C), displayed high cell viability after 14 days of preservation. The superiority of supercooling preservation at -4°C was demonstrated by comparing the obtained results with that of refrigerated preservation at +4°C. Cells preserved for 14 days under optimal conditions showed no cell shape abnormalities and may be used for experiments immediately after thawing. The optimized supercooling preservation method determined in this study is suitable for the temporary preservation of adherent cultured cells.

Feline ovarian tissue vitrification: The effect of fragment size and base medium on follicular viability and morphology.

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 × 0.75 mm and 3 × 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol®; HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL® TOTAL® LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 ± 2.0%) compared to the larger fragments (40.3 ± 2.0%). Our results demonstrated that 1.5 × 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 ± 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate.