Abstract:Spontaneously formed spheroids from mouse compact bone-derived cells (CBDCs) possess enhanced stemness and superior plasticity. Here, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CBDCs spheroids were investigated. CBDCs were isolated from mouse femur and tibia. Spheroids were cryopreserved with various concentrations of dimethyl sulfoxide (DMSO). After thawing, the number of living and dead cells were measured. The expressions of stem cell mark… Show more
“…3 ). 15 , 46 , 53 Fig. 1 An alternative spontaneous spheroid formation method using a culture dish possessing an almost 90-degree water contact angle.…”
Section: Mechanical Spheroids and Spontaneous Spheroidsmentioning
“…3 ). 15 , 46 , 53 Fig. 1 An alternative spontaneous spheroid formation method using a culture dish possessing an almost 90-degree water contact angle.…”
Section: Mechanical Spheroids and Spontaneous Spheroidsmentioning
“…Приймаючи до уваги той факт, що швидкість охолодження, прямо пов'язана зі швидкістю, з якою внутрішньоклітинна вода може виходити з клітин, тобто з коефіцієнтом проникності клітинної мембрани для води, в подальших дослідженнях за допомогою рівняння (7) були розраховані величини відносного об'єму СФ при заморожуванні з кріопротектором ДМСО зі швидкостями охолодження 0,5-5 град/хв (рис. 6).…”
Section: прогнозування осмотичної поведінки сфероїдів в умовах охолод...unclassified
“…Дослідження останніх років показали високу функціональну активність клітин, які було культивовано у вигляді сфероїдів, а також посилення протизапальної дії, ангіогенетичні ефекти та краще виживання після трансплантації [4][5][6]. Очікується, що у недалекому майбутньому сфероїдні культури (spheroid-based cultures) будуть більш затребувані у регенеративній медицині, ніж існуючі моношарові [7,8].…”
unclassified
“…Кінетику зміни концентрації позаклітинного розчину в процесі заморожування при розрахунках задавали аналітично шляхом апроксимації фазової діаграми плавлення розчину ДМСО у вигляді[25]: с 𝑇 -концентрація позаклітинного розчину, за якої він знаходиться в термодинамічній рівновазі з льодом при температурі Т; с 0 -вихідна (до заморожування) концентрація розчиненої речовини позаклітинного розчину; 𝑇 = -приведена температура; Tпоточне значення абсолютної температури, Tk0 -значення температури плавлення розчину. Вирішуючи рівняння(7) для визначених транспортних характеристик сфероїдів і вибраних швидкостей охолодження отримували залежності об'єму сфероїдів від температури заморожування. З урахуванням значень ступеня дегідратації робили висновок про оптимальну швидкість охолодження, спираючись на відповідну імовірність внутрішньоклітинної кристалізації при даному режимі охолодження СФ, як описано у роботах[13,26].РЕЗУЛЬТАТИ І ОБГОВОРЕННЯПротягом 21 доби культивування формувались переважно (70%) сфероїди округлої форми з діаметром 80-100 мкм.…”
Background: Three-dimensional culture systems are unique platforms for studying complex biological processes in vitro. Cell-cell and cell-extracellular matrix interactions form a communication network of biochemical and mechanical signals, bring spheroids (SP) closer to native tissues and significantly distinguish them from monolayer cultures. It is important for cell technologies to develop methods for cryopreservation of 3D cultures, that allows creating the stocks of valuable cell samples, save time and materials, and prevent the loss of cultures due to technical failures, contamination, phenotype drift and aging.
Objectives: Development of approaches to cellular spheroids cryopreservation. Determination of the permeability parameters of L929 cells spheroids at different cultivation periods for the theoretical assessment of optimal freezing regimens.
Materials and methods: We have used L929 cells, which form SPs of different diameters and can be maintained for a long time in 3D conditions. To determine the integral filtration Lp and permeability for DMSO kp coefficients for SP at different periods of cultivation, the volumetric method was used. The study of the changes in the spheroids volume in time was carried out with a confocal microscope LSM 510 META. The numerical values of the integral SF permeability coefficients were determined by approximating the experimental data on the change in the relative volume of the SP versus the exposure time in the test solution with theoretical curves calculated on the basis of a physical and mathematical model for passive mass transfer between the spheroid and the environment, provided that they coincide as much as possible. Prediction of the osmotic behavior of spheroids under cooling conditions was carried out based on the differential equation describing the kinetics of changes in the relative cell volume during extracellular crystallization of a cryoprotective solution, substituting determined values of integral permeability coefficients Lp and kp and activation energies EAL and EAk into the model equations. The kinetics of changes in the extracellular solution concentration during freezing was set analytically by approximating the phase melting diagram of the DMSO solution.
Results: The filtration and permeability for DMSO molecules coefficients in SP were determined and their significant decrease with a cultivation duration was shown. The activation energy values for the penetration of water and DMSO molecules into the SP were calculated and their dependence on the cultivation time was determined. Proceeding from the determined parameters of permeability, the dynamic of changes in the volume of SPs for different periods of cultivation at different rates of cooling was calculated.
Conclusions: The optimal cooling modes of SP from L929 cells were in theory determined: for 7 days of cultivation — 1,5-2 °C/min with cooling to -80°C and subsequent immersion in nitrogen; for 14 and 21 days of cultivation — 0.5 °C/min to -40°C and subsequent immersion in nitrogen.
“… 1 There is also precedent for cryopreserving cells that have been aggregated into organoids or microtissues. 10 , 11 , 23 Commercial manufacturers such as STEMCELL™ technologies recommended that their cryopreserved organoids be cultured for a few days and passaged a couple of times after thawing. If the organoids are damaged they will recover during the post-thaw culture.…”
The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O2 atmosphere than a 20% O2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function.
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