Inclusion Criteria: 1. Clinical diagnosis of DEB. 2. Age 12 months and older. 3. Willing and able to give consent/assent; if under the age of 18 years, guardian(s) is/are willing and able to give consent. 4. (This inclusion criterion was deleted as of Amendment 1.) 5. Confirmation of DEB diagnosis (either dominant \[DDEB\] or recessive \[RDEB\] forms) by genetic testing. 6. Able to undergo adequate anesthesia during EB-101 treatment. 7. All women of childbearing potential must have a negative urine pregnancy test and use a reliable birth control method throughout the duration of the study. 8. On stable pain medication regimen for at least 30 days prior to Screening (and through Baseline). 9. Must have at least one wound site that meets all of the following criteria: 1. An area ≥20 cm2, 2. Present for ≥6 months, and 3. Stage 2 wound defined as an open skin wound with partial thickness loss of dermis that has not extended through the dermis into subcutaneous tissue. 10. For patients with RDEB, they must have had Vyjuvek exposure by the time of screening or positive anti-C7 antibodies at baseline. 11. Patients must be willing to discontinue Vyjuvek and Filsuvez use on EB-101 treated sites until completion of the Week 24 visit. Exclusion Criteria: 1. Medical instability limiting ability to travel to the study site or undergo EB-101 treatment. 2. The presence of medical illness expected to complicate participation and/or compromise the safety of this technique, such as active infection with human immunodeficiency virus (HIV), hepatitis B, or hepatitis C. 3. (This exclusion criterion was deleted as of Amendment 3.) 4. Evidence of systemic infection. 5. Current evidence or a history of SCC in the area that will undergo EB-101 application. 6. Active drug or alcohol addiction. 7. Hypersensitivity to vancomycin or amikacin. 8. Receipt of chemical or biological investigational therapy for the specific treatment of DEB in the 3 months prior to EB-101 application. 9. Breast-feeding. 10. Inability to properly follow protocol assessments and protect keratinocyte sheet sites as determined by the PI. 11. Grade 3 clinical event or laboratory abnormality at Day 0. Abnormalities such as esophageal strictures, anemia, low albumin, and pain/itch are expected in severe DEB patients, and these abnormalities will not exclude a patient. 12. Unwillingness or inability to provide 4 skin biopsies, or patient's keratinocytes cannot be manufactured for use in EB-101 application. 13. Any other circumstance where the PI believes that the patient may not be appropriate for participation in the study.