Inclusion Criteria:

1. Adults 18 years and older

2. Anterior or posterior chronic anal fissure (CAF) - chronicity defined as presence of anal fissure ≥ 6 weeks

3. Inadequate response to medical treatment of anal fissure (1 month of failed vasodilator treatment plus declined or failed botulinum injection treatment)

4. Recent history of pain on defecation at a level 4 or higher on the numerical rating scale (NRS)

5. Vital signs upon screening:

   • Blood pressure: systolic ≥ 90 and < 140; and diastolic ≥ 60 and < 90.
   • Breathing: ≥ 12 and ≤ 20 breaths per minute.
   • Pulse: ≥ 60 and ≤ 100 beats per minute.
   • Temperature: ≥ 97.8°F and ≤ 99.1°F (36.5°C to 37.3°C)
   • O2 saturation: > 92%

6. Willing to take adequate contraceptive measures

7. Willing to sign an informed consent form and follow instructions for the trial including appearing for visits and filling out questionnaires

Exclusion Criteria:

1. Lateral anal fissure

2. Presence of peri-anal or rectovaginal fistula, rectal or anal stenosis, or peri-anal abscess or non-healing peri-anal post-surgical wounds that are not anal fissures (subjects with history of anorectal surgery with healed surgical wound is not excluded)

3. Active, untreated or medically unresponsive infection of the anal fissure or fistula (e.g., erythema and pus)

4. Active systemic infection (e.g., bacteremia, sepsis) - stable, controlled and treated HIV+ subjects (e.g., recent plasma HIV RNA <200 copies/mL) are not excluded

5. Presence of inflammatory bowel diseases (e.g., Crohn's, ulcerative colitis)

6. Taking systemic chemotherapy or local pelvic radiation treatments

7. Renal impairment defined by serum creatinine ≥ 1.5 x upper limit of normality (ULN)

8. Hepatic impairment defined by both of the following laboratory ranges:

   (a) total bilirubin ≥ 1.5 x ULN unless benign congenital hyperbilirubinemia; and (b) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≥ 2.5 x ULN

9. Active alcohol or substance use that, in the opinion of the site investigator, will interfere with study follow-up.

10. Active malignant tumor (tumors must be in remission for ≥ 6 months without maintenance chemotherapy and/or radiation)

11. Ongoing or recent history (within 6 months) of abnormal, severe, progressive, or uncontrolled hepatic, hematological, gastrointestinal, endocrine, pulmonary, cardiac, neurological, psychiatric, or cerebral diseases

12. Congenital immunodeficiencies

13. History of major surgery or severe trauma within the previous 3 months

14. Subjects who are actively being considered as candidates for solid organ transplantation or who may have a high likelihood of needing a solid organ transplant (ex. Progressive heart failure)

15. Females who are pregnant or breastfeeding or planning to become pregnant or breastfeed during the study (180 days)

16. Subjects who have known hypersensitivity or documented allergy to DMSO

17. Subjects who do not wish to or cannot comply with study procedures