Liver cirrhosis is the eventual outcome of chronic liver diseases, including chronic hepatitis, autoimmune hepatitis, primary biliary cholangitis, alcoholic hepatitis, and nonalcoholic steatohepatitis (NASH), which is currently increasing in prevalence relative to other liver conditions and is associated with metabolic disease [1,2]. Liver cirrhosis leads to various complications, including esophageal varix, hepatic encephalopathy, ascites, jaundice, and hepatocellular carcinoma, which result in a worse prognosis for patients with cirrhosis [3]. The end stage of cirrhosis is hepatic failure, for which liver transplantation is the only treatment as an extremely invasive procedure that requires continuous immunosuppressive therapy for the remainder of the patient’s life. Moreover, there are very few cadaveric donors in Japan, with only 69 cadaveric donors (and 347 living donors) for liver transplantation available in 2017 [4]. Therefore, novel treatments for cirrhosis should be explored.

Fatty liver disease and NASH are emerging chronic liver diseases. Although steatohepatitis is strongly correlated with metabolic syndrome, it is not clear why steatosis of the liver causes chronic inflammation and ultimately leads to cirrhosis [5].

Mesenchymal stem cells are somatic pluripotent stem cells that can differentiate into various cell types [6,7] and have immunomodulatory capabilities [8]. Freshly isolated adipose tissue is a rich source of mesenchymal stem cells [9]. Similar to bone marrow cells, autologous mesenchymal cells can be used therapeutically. Therefore, freshly isolated autologous adipose tissue-derived stromal cells have attracted substantial attention for potential therapeutic use in various organs [10]. We previously conducted a clinical study to confirm the safety of a liver cirrhosis therapy based on adipose tissue-derived stromal cells [11]. To determine the practicality of this treatment strategy, we designed an open-label, uncontrolled multi-institutional clinical trial for a regenerative therapy for liver cirrhosis based on adipose tissue-derived regenerative (stem) cells (ADRCs). This trial was designed to assess the safety and efficacy of the treatment in consultation with the Japan Pharmaceuticals and Medical Devices Agency (PMDA). Herein, we describe the detailed protocol for the trial.

The objective of the study is to assess the efficacy and safety of an autologous ADRC therapy delivered to the liver via the hepatic artery.

This is a multicenter, collaborative, nonblinded, uncontrolled clinical trial carried out in Japan (Kanazawa University Hospital, Osaka Medical College Hospital). The adipose tissue dissociation device used to isolate ADRCs is the investigational trial device. All patients who meet the eligibility criteria and provide informed consent will be enrolled (Figure 1). Multimedia Appendix 1 shows the detailed schedule of the clinical study.

Subcutaneous adipose tissue from the abdomen or buttock of the patient will be obtained by Tumescent liposuction, a standard cosmetic surgery method, under general anesthesia or local/lumbar spine local anesthesia. Regenerative (stem) cells will be isolated using the adipose tissue dissociation system Celution 800/IV (Cytori Therapeutics, San Diego, CA, USA), followed by measurements of cell number and viability in a collected cell aliquot using a nucleocounter (ChemoMetec, Gydevang, Denmark). Cell viability should be at least 70%. Cell aliquots will be prepared at a density of 1×10⁶/mL in Ringer’s lactate. ADRCs, at a density of 3.3×10⁵ cells/kg, will be administered through the common hepatic artery via the tip of microcatheter IV (Asahi Intec Co Ltd, Seto, Aichi, Japan), inserted subcutaneously into a femoral or upper arm artery. For 24 weeks after providing informed consent, albumin products, antiplatelets, and anticoagulants will be prohibited. The following drugs can be continued only when administered after informed consent has been provided: branched-chain amino acids, vitamin E, other nutritional therapies, hepatic protection drugs, and antihyperlipidemia drugs.

Abdominal computed tomography or magnetic resonance images of the liver will be obtained using a contrast reagent before registration and 24 weeks after treatment. These images will be analyzed by a radiologist and hepatologist who will assess the morphological changes in the liver, spleen, and vasculature due to chronic liver disease. The pathology will be assessed using the nonalcoholic fatty liver disease activity score, which covers steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis [14], in addition to the Matteoni classification [15].

The observation period after treatment will be 24 months during which examinations will be performed at various time points (Multimedia Appendix 1).

Efficacy endpoints are the serum albumin level and prothrombin activity. The safety endpoints are adverse reactions and device malfunction.

The serum albumin level is considered a key efficacy endpoint with respect to the proof of concept and mode of action of this treatment. In an earlier clinical study, we observed that 3 of 4 cirrhotic patients who underwent the same treatment as applied in this clinical trial had improved serum albumin levels after 3-6 months [11]. A sample size of 7 patients provided a power of over 85% with a one-sided significance level of .05 to reject the null rate of 3% under the expectation of 40%. This clinical trial was designed to explore safety and efficacy, and therefore a total of 8 patients was considered to be sufficient for this purpose.

A detailed statistical analysis of efficacy will be performed, and a per-protocol analysis will also be conducted secondarily. Subjects who underwent treatment will be used for the safety analysis. The statistical analysis protocol will be designed before data confirmation and fixation.

Approval of this clinical trial was obtained from the institutional review boards of Kanazawa University Hospital and Osaka Medical College Hospital. The study has been registered in the Japanese UMIN Clinical Trials Registry (UMIN000022601). This investigator-initiated clinical trial design was discussed with, and submitted to, the Japan PMDA before enrollment commenced. All treatment procedures in this trial will be carried out in accordance with the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice guidelines [16]. Written informed consent will be obtained from the patients before the first and second screenings as well as at registration.