Dispase

Manufactured by Boehringer Ingelheim

Sourced in United States

Dispase is a proteolytic enzyme that can be used to dissociate adherent cell layers from a variety of substrates. It exhibits broad specificity for cleavage of extracellular matrix proteins.

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Lab products found in correlation

5 protocols using dispase

Establishing Primary Corneal Epithelial Cultures

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All work with human tissue was done in accordance with the tenets of Helsinki. Donor human corneoscleral rims with no history of corneal diseases that were unsuitable for transplant were used to establish primary epithelial cultures as previously described [9] (link), [38] (link). Human cadaver corneas were graciously donated by the Lions Eye Bank of Wisconsin, Madison or the Missouri Lions Eye Bank (Columbia, MO). These tissue samples obtained were not human subjects research and as such approval from committee or kin were not necessary. Briefly, sclera and limbal regions of the cornea were trimmed and the tissue was immersed in dispase (1.2 U/ml, Boehringer, Mannheim, Germany) at 37°C for 4 h. Corneal epithelial cells were removed by gently rubbing the anterior surface with a sterile pipette tip. Cell suspensions were pooled, centrifuged and re-suspended in EpiLife medium (Life Technologies, Carlsbad, CA) supplemented with EpiLife defined growth supplement (EDGS; Life Technologies) and 1% penicillin/streptomycin (Life Technologies) and used between passages 2 and 3. Experiments were repeated with cells isolated from three different donors.
Immortalized human corneal epithelial cells (hTCEpi; [44] (link)), kindly provided by Dr James V Jester (UC Irvine), were maintained in EpiLife medium as above and were used between passages 40–60.

Raghunathan V.K., Dreier B., Morgan J.T., Tuyen B.C., Rose B.W., Reilly C.M., Russell P, & Murphy C.J. (2014). Involvement of YAP, TAZ and HSP90 in Contact Guidance and Intercellular Junction Formation in Corneal Epithelial Cells. PLoS ONE, 9(10), e109811.

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Keratinocyte Isolation and Culture

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Skin biopsies for keratinocyte cultures were voluntarily provided by patients undergoing routine operations with skin graft procedures, and informed consent was obtained from all subjects (ethics approval Nr. 264_13 B Friedrich Alexander University Erlangen-Nürnberg). Proliferative human keratinocytes were isolated from fresh human skin with 0.25% Dispase (Boehringer, Mannheim, Germany) after incubation at 40°C for 2 h. Epidermal cells were then isolated into a single-cell suspension by treatment with 0.05% trypsin and 0.02% EDTA (Gibco, Germany) at 37°C for 30 min, resuspended, and expanded in 75 cm² polystyrene tissue culture flasks in serum-free media containing EGF, BPE (Gibco, Germany), and gentamycin (Merck, Germany) at 5% CO₂ and 37°C. At 60–70% subconfluence, the cells were then harvested from the flasks by trypsinization and then re-incubated in a concentration of 1×10⁶ per 75 cm² culture flask [4 (link)].

Horch R.E., Wagner G., Bannasch H., Kengelbach-Weigand A., Arkudas A, & Schmitz M. (2019). Keratinocyte Monolayers on Hyaluronic Acid Membranes as “Upside-Down” Grafts Reconstitute Full-Thickness Wounds. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 6702-6710.

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Dental Pulp Stem Cell Isolation

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Human exfoliated deciduous incisors were obtained as discarded biological samples from 6-to-8-year-old children in the Hospital of Stomatology, China Medical University, per the Institutional Review Board guidelines. Informed consent was obtained from the children's parents. The pulp was separated from the exfoliated teeth and digested in a solution of 4 mg/mL dispase (Boehringer Ingelheim, Mannheim, Germany) and 3 mg/mL collagenase type Ι (Worthington Biochemical Co., Lakewood, CO, USA) at 37°C for 30 min. Single-cell suspensions were obtained by passing the digest through a 70 μm strainer (Merck Millipore Ltd., Cork, Ireland). The cells were cultured in alpha modification of Eagle's medium (HyClone, Logan, UT, USA), 15% fetal bovine serum (ExCell Bio, Shanghai, China), 100 mmol/L L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and 100 U/mL penicillin-streptomycin (HyClone) at 37°C in the presence of 5% CO₂. SHED of passages 3–5 were used in the experiments. Serum-free culture medium was used as the cell suspension medium for transplantation.

Bai X., Zhang X., Wang C., Liu Y., Liu X., Fan Y, & Zhang X. (2021). Stem Cells from Human Exfoliated Deciduous Teeth Attenuate Trigeminal Neuralgia in Rats. Stem Cells International, 2021, 8819884.

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Isolation and Culture of hPDLSCs

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According to the protocol approved by the Institutional Review Board (IRB No. WKDIRB-201708-02) of the dental clinic of Wonkwang University, the third human molars were collected from three healthy young females (15–23 years old). After gently separating the PDL from the extracted molar, the separated PDL was digested in 3 mg/mL collagenase type I solution (Worthington Biochem, Freehold, NJ, USA) and 4 mg/mL dispase (Boehringer, Mannheim, Germany). For single-cell suspension, whole cells were passed through the 40-mm strainer (Falcon BD Labware, Franklin Lakes, NJ) and were incubated in alpha-modified Eagle's medium (αMEM; Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; Gibco BRL) and 1% penicillin-streptomycin antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) in a 37 °C, 5% CO₂ incubator. The medium was changed after the first 24 h and every three days thereafter. Colonies of hPDLSCs were randomly selected and incubated separately. All primary cells within passage 2 or 3 were used in this study.

, & Han Y. (2021). High concentrations of calcium suppress osteogenic differentiation of human periodontal ligament stem cells in vitro. Journal of Dental Sciences, 16(3), 817-824.

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Isolation and Characterization of Human PDL Stem Cells

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Non-decayed human 3rd molars that had been impacted in the mandible were extracted from 5 adults (18–28 years of age) with informed consent at the Seoul National University Dental Hospital, Seoul, South Korea. The experimental protocol was approved by the Institutional Review Board of the hospital (IRB No. 05004). The PDL was gently separated from the root of the extracted 3rd molars, and the separated tissues were digested in a solution of 3 mg/mL collagenase type I (Worthington Biochem, Freehold, NJ, USA) and 4 mg/mL dispase (Boehringer, Mannheim, Germany) for 1 h at 37 °C. Single-cell suspensions were collected by passing the cells through a 40 mm strainer (Falcon BD Labware, Franklin Lakes, NJ, USA) and were cultured in the alpha-modification of Eagle’s medium (α-MEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco BRL), 100 mM ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Biofluids, Rockville, MD, USA). The medium was changed after the first 24 h and then every 3 d. A total of 3 colonies of hPDLSCs were randomly picked, and the cellular pool of these colonies was used for in vitro proliferation, differentiation studies, and animal experiments. All primary cells used in this study were in cell passage stages 2 or 3.

Lee U.L., Yun S., Cao H.L., Ahn G., Shim J.H., Woo S.H, & Choung P.H. (2021). Bioprinting on 3D Printed Titanium Scaffolds for Periodontal Ligament Regeneration. Cells, 10(6), 1337.

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