What is HUCT?
Human Umbilical Cell Tissue is stem cells extracted from the umbilical cord, placenta and cord blood from healthy mothers who have been prequalified and deliver through a c-section. HUCT are captured at the earliest point the umbilical cord, placenta and cord blood are ready for utilization in its earliest stages making them some of the best stem cells available for therapy purposes.
Using HUCT stem cells aids in healing otherwise deficient human cells, tissues and organs. In regenerative medicine, the use of cell replacement approaches which usually requires stem cells is a primary therapy. Current research and scientific studies have shown that stem cells can replace bone, fat, cartilage, heart tissue and muscle. Use of stem cells from HUCT has much potential for helping to heal or reduce the severity of many disease states. The umbilical cord serves as a conduit of nutrients for a fetus. Oxygenated nutrient rich blood is carried from the placenta to the fetus until the baby is born. This cord blood within the tissue is rich in primitive stem cells, growth factors and immune cells that are nave as they have to be compatible for the baby and mother. Moreover, the use of allogeneic cord blood has been used for decades in greater than 500 patients with diseases such as Hurlers syndrome, Duchenne muscular dystrophy and Krabbes disease. Most importantly it is safe. These otherwise discarded tissues are an excellent source of high-quality stem cells and growth factors which may be used in regenerative medicine. Utilizing HUCT from the newborn after birth increases the potency and effectiveness of the cells because it is a known fact as we age the number of stem cells greatly diminishes making the HUCT protocol a very popular solution.
Stem cells that are a derivative from umbilical cord tissue are clinically the least invasive and safest method of removal available. Alternative options include extraction from embryonic tissue that is derived from embryos, bone marrow which can be extracted by aspiration and is usually painful, and adipose tissue which can be surgically extracted through liposuction under general anesthesia.
The human umbilical cord is a reliable source of mesenchymal stem cells (HUCMSC). Unlike bone marrow stem cells, HUCMSCs have a comparatively painless collection procedure and faster self-regenerative properties. Different derivation protocols may provide different amounts and populations of stem cells. Stem cell populations have also been identified in other compartments of the umbilical cord, such as the cord lining, perivascular tissue, and Whartons jelly. The use of HUCMSCs are noncontroversial sources compared to embryonic stem cells. They can differentiate into the three germ layers that promote tissue repair, moderate immune response, and anti-cancer properties. Additionally, they are considered more beneficial autologous or allogeneic agents for the treatment of malignant and nonmalignant solid and soft type cancers. Other alternative benefits from HUCT include correction of corneal epithelial defects, burn and diabetic wound ulcer treatment modalities, better osteogenesis capabilities, rescue of liver fibrosis, and hyperglycemia in the diabetic population. In comparison, embryonic stem cells (ESCs) can differentiate into almost all tissues in the human body and are thus labeled as pluripotent. However, the use of ESCs generated from embryos has raised ethical concerns. Furthermore, the clinical applications of therapies derived from embryonic stem cells have been criticized because of the possibility of forming tumors by integrated oncogenes and suppression of cells that disrupt tumor formation. This alternative portrays HUCT as a better, safer choice for clinical applications, efficiency, and safety precautions. Regarding the therapeutic principles of HUCT, effective storage banking systems and protocols should be established immediately.
Isolation by enzymatic digestion Type I collagenase, or collagenase type A, is extensively used for the isolation of mesenchymal-like cells from the cord tissue to facilitate the degradation of matrix ground substance and shortens the time required for the isolation process. The time required for tissue digestion ranges from 30 minutes to 16 hours depending on the quantity/concentration of enzyme and duration of treatment with digesting reagents. Filtration of the digested material through 70100m pore sized cell strainers facilitates the removal of any unwanted tissue debris. Isolation by explants culture The principle of the method is generally described as fine chopping of the Whartons jelly sections of the cord tissue, after excision of the blood vessels, with a scalpel, plating of the fine fragments in sterile culture plates or Petri dishes, and culturing of these with low-glucose DMEM, supplemented with foetal bovine serum (10-20% v/v), L-glutamine and antibiotics/antimycotics. Cryopreservation methods for UCT and hMSCs isolated from UCT: Slow cooling and Vitrification Cryopreservation of cord tissue and/or cells extracted from the tissue represents an important stage to overcome in the view of the therapeutic use of stem cells. HUCT cryopreservation should be able to maintain the cellular metabolism in a dormancy state for an indefinite period of time. Use of defined culture media supplemented with high amounts of foetal bovine serum and 7% 10% (v/v) dimethyl sulfoxide (DMSO) or glycerol and freeze the cells gradually (eg. 10C/min) and keep them between -135C and -196C. After rapid thawing at 37C, viability rates of over 50% can be achieved.
The samples should be frozen for a period of time ranging from 5 to 78 days. The slow cooling protocol was more efficient than the vitrification, for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro is greatly preserved. Cryopreservation of small fragments of tissue from the umbilical cord and to obtain viable cells capable of proliferation in vitro after thawing, contribute to the creation of a frozen tissue bank. In vitro differentiation To successfully differentiated lineages using a variety of cell culture techniques and reagents for in vitro differentiation of Adipocytes, Chondrocytes, Osteocytes, Cardiomyocytes, Skeletal myocytes, Neuronal/glial precursors, Dopaminergic neurons, and Endothelial cells. Independently of their origin, the adipogenic potential of HUCT MSCs is inversely related to the length of in vitro culture, and sharply declines when HUCT MSCs become senescent. Contrary, prolonged culturing increases their osteogenic differentiation. In vitro expansion of HUCT MSCs should, therefore, be performed with limited passaging, to avoid changes in their differentiation ability. Gradual shortening of the telomeres during a cells life continues until the presence of critically short telomeres triggers a senescence pathway, which results in proliferation arrest. Because of that, a normal human cell can only divide 50 to 100 times in vitro conditions; HUCT MSCs are no exception. UC blood HUCT MSCs, however, have slightly longer telomeres than other MSCs, and thus can be cultured for longer periods before they senesce. Proliferation arrest in HUCT MSCs results in their senescence, which is described by the appearance of large senescent cells with a flat shape, circumscribed nuclei, and increased lysosome compartment. These morphological changes are not restricted to the senescent stage only, but represent continuous alterations in the course of HUCT MSCs long-term culture. Immunophenotyping of HUCT MSCs Characterisation of HUCT MSCs is generally accomplished by flow cytometry analysis of surface markers. Stro-1 has been identified as a marker for cells that can differentiate into multiple mesenchymal lineages. CD9/CD90/CD166 triple positive subpopulation of HUCT MSCs showed multipotency for chondrogenic, osteogenic and adipogenic differentiation providing a basis for identification of HUCT MSCs. It has been indicated that positive expression of CD166 is indicative of multipotency in HUCT MSCs. Expression levels of CD90 and CD105 are maintained over sequential passages and they can be important for validating cultures of HUCT MSCs intended for therapy. A good indication of HUCT MSC identity can be reached by expression of CD90, CD105 and CD166 and lack of expression of CD34 and CD45 as a minimum set of surface markers. Variability in MSCs extraction from HUCT There are ways of minimizing variations between lots produced, by controlling process parameters, and by screening the raw materials that will be in contact with the cells and cell source. There are other noncontrollable parameters such as the source of the cells, which represents a real challenge for regenerative medicine applications. Each cell extraction method is produced with cells from a different patient/donor, with intrinsic characteristics that result in variations of cell growth patterns and differentiation. It is, therefore, necessary to develop extraction methods that are suitable to real world applications. It is necessary to map the operating environment and assess risk factors before empirically determining the effect on the process. This will be particularly critical for processes using primary tissue or cell sources where the biological variation at input is likely to be high. Regulated therapeutic products will require characterized and risk assessed manufacturing processes. This fits the philosophy of process control industry tools such as quality by design (QbD) and Six Sigma, represent approaches to understanding process operating space and risks of associated variables. Extraction of hMSCs from umbilical cord tissue via enzymatic digestion. 200-400 mg cord slices from multiple cords, both fresh and frozen, with the purpose to screen different methods for an assessment of method success with a view to downstream standardization of the isolation and expansion of mesenchymal stem cells from the HUCT aided in the development of this protocol.
2 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 40 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue is kept frozen through this initial process.
4 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 70 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue still remains frozen.
18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 70 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue still remains frozen.
2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 18 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 70 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue is fresh (2 days old).
2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 18 (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers and centrifuged at 500 rcf for 10 min/each; cord tissue is frozen.
2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers; no centrifugation for slices digested with 2 of the enzymatic solutions and 1000 rcf centrifugation speed for slices digested with the 3rd type of enzymatic solution; cord tissue is frozen.
2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers; no centrifugation for slices digested with 2 of the enzymatic solutions and 1000 rcf centrifugation speed for slices digested with the 3rd type of enzymatic solution; cord tissue is fresh (not frozen).
18 hours enzyme digestion of cord tissue with cord banks method and reagents: (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers and diluted with 3ml of culture media, before being plated in T25 flasks; no centrifugation; cord tissue is frozen.
a) Vials containing cryopreserved 200-400 mg cord tissue slices are defrosted by placing them in a 37C water bath for 3-5 minutes, or until only a trace of ice remains b) After, cryovials are transferred to a Class II biological safety cabinet (BSC) and the cord sections are removed from cryovials with a sterile aspirator stripette and placed in a Petri dish containing DPBS + 1% antibiotic/antimycotic v/v (PSA) in it. c) Individual slices are transferred to a fresh, sterile Petri dish, and chopped up into fine fragments (1-2mm3 ), with the aid of a scalpel and forceps. The fragments are then placed into a 15 ml centrifuge tube, with the aid of a scalpel and forceps. d) Cord slice fragments are enzymatically digested for 2h, 4h or 18h at 37C, with the following enzymatic solutions: A. Collagenase type I, (in serum free growth Medium A, 3-5ml/slice), 300 CDU/ml; B. Collagenase type I, 300 CDU/ml + hyaluronidase, 1mg/ml (in serum free growth Medium A, 3-5ml/slice); C. Collagenase type I, 300 CDU/ml for 1, 3 or 17 1/2 h, depending on the digestion period, followed by trypsin-EDTA 0.25% for a further 30 min (both enzyme solutions are prepared in serum free growth Medium A, 3-5ml/slice) e) Upon completion of digestion, tubes containing slices digested with enzymatic solutions A and B, are treated as follows: 5.1 Diluted 50% with serum free growth Medium A. 5.2 Filtered through a 40m, a 70m, or a 100m cell strainer; squeezing remaining tissue fragments with the forceps to aid cell release after filtration. 5.3 Centrifuged cell suspension at 1500 rcf for 10 min, 500 rcf for 10 min, or no centrifugation. 5.3.1 In the case of no centrifugation, an appropriate amount of FBS (final concentration 10%) os added to the suspension before filtration and 2ml of fresh growth media to wash the cell strainer with. 5.3.2 For methods that involved centrifugation the supernatant is discarded and the pellet re-suspended in 5ml of fresh growth Medium A; 5.4 Count cells by using a disposable haemocytometer (20l of cell suspension and 20l of trypan blue); and seeded at 104 cell/cm2 , in an appropriately sized culture vessel. f) Upon completion of digestion, tubes containing slices digested with method C, are treated according to the protocol below: 6.1 Diluted 50% with serum free growth Medium A.Centrifuged at 1500 rcf for 10 min, 500 rcf, or 1000 rcf. 6.2 Discarded supernatant and re-suspended pellet in 3-5ml of 0.25% trypsin-EDTA; 6.3 Replaced in the incubator for another 30 minutes. 6.4 After 30 min took tubes out and added 0.3-0.5ml of FBS/each tube to stop the enzyme action. 6.6 Diluted 50% with serum free growth Medium A. 6.5 Filtered through a 40m, a 70m, or a 100m cell strainer; squeezing remaining tissue fragments with the forceps to aid cell release after filtration. 6.7 Centrifuged at 1500 rcf for 10 min, 500 rcf, or 1000 rcf. 6.8 Discarded supernatant and re-suspended pellet in 5ml of fresh Medium A. 6.8.1 Counted cells by using a disposable haemocytometer (20l of cell suspension and 20l of trypan blue); and seeded at 104 cell/cm , in an appropriately sized culture vessel. g) All culture vessels are incubated at 37C and 5% CO2 in a humidified incubator. h) First media change is performed after 48h and every 3 days thereafter until cells have reached 80-85% confluence.
Protocol for enzymatic digestion of fresh cord tissue Procedure: a. For processing, cord sections are removed from tubes, inside a BSC, with sterile forceps and positioned on sterile prep trays; the outside of the cord is wiped with alcohol wipes (also held with sterile forceps to avoid touching cord surface). The remaining cord blood is squeezed from the cord by pressing the blunt edge of a sterile scalpel along the length of the cord. b. The cord tissue sections are then placed in a Petri dish with DPBS and 1% PSA in it. Swirled contents to wash. If the saline water is really cloudy with blood, the wash step is repeated. c. Cord sections are cut into 200-400 mg slices (approximately 2-4mm thick, depending on the thickness of the cord), and placed into separate Petri dishes with fresh DPBS and 1% PSA, to wash. The slices are then placed in separate Petri dishes with warm, serum free Media A. Each slice is weighed in a pre-weighed sterile, closed container; only slices that weigh approximately 300 mg are used, in order to maintain consistency. d. Each slice, is placed on a separate, sterile Petri dish, and chopped up in fine fragments (1-2mm). The fragments from each slice are placed in individual 15 ml centrifuge tubes. e. Cord fragments are enzymatically digested for 2h, 4h or 18h at 37C, with the following enzymatic solutions: A. Collagenase type I, (in serum free growth Medium A, 3-5ml/slice), 300 CDU/ml; B. Collagenase type I, 300 CDU/ml + hyaluronidase, 1mg/ml (in serum free growth Medium A, 3-5ml/slice); Mesenchymal Stem Cell E, C. Collagenase type I, 300 CDU/ml for 1, 3 or 17 1/2 h, depending on the digestion period, followed by trypsin-EDTA 0.25% for a further 30 min (both enzyme solutions being prepared in serum free growth Medium A, 3-5ml/slice); f. For tubes containing slices digested with methods A and B, after digestion time had finished, refer to previous protocol, step e-g. For tubes containing slices digested with method C, after digestion time has finished refer to previous protocol, step f. g. All culture vessels are incubated at 37C and 5% CO2, in a humidified incubator. h. First media change is performed after 48h and every 3 days thereafter until cells reached 80-85% confluence.
Protocol for enzymatic digestion of fresh and frozen cord tissue with the cord banks method a. Take each UCT slice, placed on separate, sterile Petri dish, and chop it up into fine tissue fragments (1-2mm3 ), which are then placed in individual 15 ml centrifuge tubes; b. Fragments from each slice are digested for 18h with: 2.1 collagenase type I (AMS Biotechnology Ltd, UK) 5ml/slice/tube; enzyme solution is prepared in serum free growth Medium B, at a concentration of 0.075% (750 CDU/ml) enzymatic solution A (used by cord blood bank); 2.2 collagenase type I (Sigma Aldrich, UK) 5ml/slice/tube; enzyme solution is prepared in serum free growth Medium B, at a concentration of 0.075% (750 CDU/ml) enzymatic solution B. c. Upon completion of digestion: Filter all digested slices through 100m cell strainer in 50 ml tubes; squeezing remaining tissue fragments with the forceps to aid cell release after filtration. 0.5ml FBS (provided by cord blood bank) + 3ml growth Media B are added to the suspension resulted from a slice digested with enzymatic solution A, through the cells strainer; this action served two purposes, releasing the remaining cells on the strainer and dilution of suspension. d. 0.5ml FBS (provided by cord blood bank) + 3ml growth Media B is added to the suspension, resulted from a slice digested with enzymatic solution B, through the cells strainer. 0.5ml FBS + 3ml growth Media B is added to the suspension resulted from a slice digested with enzymatic solution A, through the cells strainer. 3.3 The cell suspension obtained after filtration and dilution is seeded in T25 culture flasks. e. All culture vessels incubate at 37C and 5% CO2, in a humidified incubator. f. After 48h, culture flasks are removed from the incubator, spent media containing dead cells and extracellular matrix, left over from the digestion process, is aspirated and a wash with warm DPBS is performed. After washing the surface of the cell culture, fresh, warm (37C), growth Media B is added. Media change was performed after that every 3 days until cells reached 80-85% confluence.
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