Methods and devices for enhanced recovery of cells and cell-rich matrices from tissue samples
A tissue sample and tissue technology, applied in cell dissociation methods, bone/connective tissue cells, biomaterial sampling methods, etc., can solve problems that have not been fully elucidated
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Embodiment 1
[0118] Fresh dog omental adipose tissue was obtained from tissue discarded after neutering surgery. Cut the tissue into pieces with sterile scissors, and then aliquot (approximately 2g / tube) into 50mL sterile centrifuge tubes. Sterile lactated Ringer's solution containing a blend of bacterial collagenases I and II together with dispase was added, and the tubes were randomly assigned to either incubate on a shaker (60 rpm) or to in a stationary rotor. Heated tissue processing apparatus (TPA, 3 cycles per minute from 1x g to 200x g to 1x g). The temperature was maintained between 37-40°C and the incubation / treatment was carried out for 30 min.
[0119] After treatment, the dissociated tissue slurry was passed through a 100 μm filter and the cell fraction was recovered from the filtrate by centrifugation in TPA at 400×g for 10 min. Cell fractions were plated on 25 cm 2 In tissue culture flasks, grow for 2 days at 37°C in DMEM / 20% (v / v) fetal bovine serum (FBS) containing antib...
Embodiment 2
[0121] Fresh human lipoaspirate was obtained from patients undergoing elective liposuction with informed patient consent. Tissue was drained using a sterile stainless steel filter and aliquoted (approximately 10 g / tube) into a tube with (see Figure 3A , 3D and 3E) or in sterile 50 ml centrifuge tubes without inserts made of nylon mesh with a pore size of 1 mm. Sterile Lactated Ringer's solution containing a blend of bacterial collagenases I and II together with dispase was added and the tubes were randomly assigned to incubate in a shaker (60 rpm) or in a swinging bucket rotor heated TPA (3 cycles per minute from 1x g to 200x g to 1x g). The temperature was maintained between 37-40°C and the incubation / treatment was carried out for 30 min.
[0122] After treatment, the dissociated tissue slurry was passed through a 100 μm filter and the cell fraction was recovered from the filtrate by centrifugation in TPA at 400×g for 10 min. Cell fractions were plated on 25 cm 2 In tis...
Embodiment 3
[0124] Lipoaspirate samples were obtained from human patients undergoing elective liposuction. Lipoaspirate was transferred to multiple tissue collection containers with a volume of 20 cc. The lipoaspirate contents of each tissue collection container were expressed 5 times through a microemulsification needle. Squeezed lipoaspirate from each tissue collection container was then transferred to a separate tissue collection container. The tissue collection container is then placed in the lumen of the removable carousel within the automated tissue processing unit. The removable swivel mechanism allows the tissue collection container to maintain a horizontal position while the automated tissue processing unit applies acceleration and centrifugal force to the contents of the tissue collection container. Centrifugal force is applied at a rate of about 400 x g, about 700 x g, about 1200 x g for 30 minutes, or at a rate of about 2000 x g for 30 minutes. For each centrifugal force ra...
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