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Therapeutic apheresis
Published in Jennifer Duguid, Lawrence Tim Goodnough, Michael J. Desmond, Transfusion Medicine in Practice, 2020
Differential centrifugation has historically been the technology principally used in apheresis. With differential centrifugation, blood enters a rotating bowl, chamber, or tubular rotor, or a belt-shaped channel, and as the blood settles in the centrifugal field, blood components layer out based on relative density. The densest cells are the red blood cells, which are concentrated in the area where the gravitational forces are greatest, followed by granulocytes, mononuclear cells (including lymphocytes and peripheral blood progenitor cells), platelets, and plasma.
Hyaluronic Acid, Platelet-Rich Plasma, and Polylactic Reasorbable Threads in Acne Scars
Published in Antonella Tosti, Maria Pia De Padova, Gabriella Fabbrocini, Kenneth R. Beer, Acne Scars, 2018
Gabriella Fabbrocini, Marianna Donnarumma, Maria Vastarella
PRP is obtained from a sample of patients' blood drawn at the time of treatment. A 40 cc venous blood draw will yield 7–9 cc of PRP depending on the baseline platelet count of an individual, the device used, and the technique employed. The blood draw occurs with the addition of an anticoagulant, such as acid citrate dextrose (ACD) to prevent platelet activation prior to its use. PRP is prepared by a process known as differential centrifugation. In differential centrifugation, acceleration force is adjusted to sediment certain cellular constituents based on different specific gravities.
Experimental Models for Studying the Interaction of Kupffer Cells and Hepatocytes
Published in Timothy R. Billiar, Ronald D. Curran, Hepatocyte and Kupffer Cell Interactions, 2017
B.G. Harbrecht, T.R. Billiar, R.D. Curran
Both parenchymal cells and NPC will be present in the cell suspension produced by the above steps. The remaining steps of the procedure involve purification of the parenchymal cell population. Differential centrifugation is the simplest method for separating HC from NPC and takes advantage of the greater density of HC compared to NPC. A series of short, low-speed (50 g) centrifugations is performed to sediment most if not all of the heavier HC, thus establishing the fact that the purity of the HC isolation will be inversely proportional to HC yield.7 The use of metrizamide, percoll, or dextran-poly-ethylene glycol gradients have also been applied to purifying suspensions of HC.7,16,17 Coating of the culture plates with gelatin or collagen prior to plating the purified HC suspension promotes attachment and adherence.7 The standard culture media and its added supplements are listed in Table 2, as previously described.15 The cells will flatten and form aggregates during the first 24 h of culture. Sufficient attachment will occur within the first 2 to 4 h of culture to allow a washing step at this point to remove nonviable or nonadherent cells if desired.18 HC may be maintained in culture for a week or more, but will have a progressive cell loss with time.11 Addition of dexamethasone or dimethyl sulfoxide to the culture media has been used to improve HC viability.19,20 It has been our practice to culture the HC for 24 h in isolation prior to further manipulations.14
Evaluation of MTBH, a novel hesperetin derivative, on the activity of hepatic cytochrome P450 isoform in vitro and in vivo using a cocktail method by HPLC-MS/MS
Published in Xenobiotica, 2021
Yan Qin, Haijun Dong, Jiayin Sun, Yilong Zhang, Jun Li, Tianci Zhang, Guanjun Chen, Sheng Wang, Shuai Song, Wei Wang, Yuru Fan, Jie Wang, Xiaohui Huang, Chenlin Shen
RLMs were prepared by differential centrifugation as reported method (Zhu et al. 2018). Rat livers were finely minced and homogenised with 4-fold volumes (w/v) of ice-cold PBS buffer. The homogenate was centrifuged at 9000 g for 20 min at 4 °C, then the resulting supernatant was ultracentrifuged at 100 000 g for 60 min at 4 °C to obtain the pink microsomal pellet. The washed pellet was resuspended in an equal volume of 50 mmol/L Tris–HCl buffer (pH 7.4, containing 20% glycerol, 1 mmol/L EDTA, 0.25 mol/L sucrose) and stored at −80°C until use. Protein concentration was measured using the bicinchoninic acid (BCA) kit (Castiblanco et al. 2018). RLMs obtained from MTBH or phenobarbital (positive group) treated rats were added to the incubation systems as described above. The incubation samples were collected and prepared as described above, and substrate metabolites were detected by HPLC-MS/MS.
Small extracellular vesicles (sEVs): discovery, functions, applications, detection methods and various engineered forms
Published in Expert Opinion on Biological Therapy, 2021
Manica Negahdaripour, Hajar Owji, Sedigheh Eskandari, Mozhdeh Zamani, Bahareh Vakili, Navid Nezafat
Indeed, traditional sEV isolation techniques and technical standardized methods suffer from several disadvantages resulting in undesirable outcomes. Recent surveys have therefore focused on improving traditional methods especially toward the establishment of nano-based techniques (Table 2). Various sEV characteristics including density, shape, size, and surface proteins are employed in traditional methods. The methods such as centrifugation, filtration, precipitation, immunoisolation, and liquid chromatography techniques fall into the traditional category [122]. Differential centrifugation is rationally the most basic method that could be used widely for the isolation of sEVs from cell-culture medium or body fluids (urine, saliva, semen, blood, and so on). The vesicle size overlapping of sEVs, microvesicles, and apoptotic bodies prevents sEV isolation from microvesicles and apoptotic bodies through differential centrifugation [123].
Promising RNA-based cancer gene therapy using extracellular vesicles for drug delivery
Published in Expert Opinion on Biological Therapy, 2020
Vivian Weiwen Xue, Sze Chuen Cesar Wong, Guoqi Song, William Chi Shing Cho
EVs show broad prospects in cancer gene therapy. However, there are still many unknowns about the function and working mechanism of EVs. Firstly, there is no standardized method for isolation, purification, and quantification of EVs. For isolation of EVs, differential centrifugation, density-gradient centrifugation, and immunoaffinity are the three most common methods. However, differential centrifugation and density-gradient centrifugation are time-consuming and do not ensure the purity of isolated EVs. Immunoaffinity provides better isolation and purification to separate subpopulations of EVs, but the process is complex and costly [57]. In addition, EVs from different cell sources or donors have significant differences in size, shape, and cargos. This increases the difficulty of quality control for EVs production. For example, mesenchymal stem cells (MSCs) and DCs are the most common sources of EVs and drug endogenous loading [11,56]. However, until now there is no effective way to control or limit the natural cargos and the efficiency of pharmaceutical packaging for EVs generated from MSCs and DCs. Besides, the preservation of EVs and therapeutic RNA is another challenge, and the temperature lower than −80°C is commonly needed to avoid degradation [51].