Methods of preparing sterile de-cellularized human amniotic fluid that is amenable for long-term storage without loss of biological functions have been developed. In particular, the methods involve refrigeration steps to maximize shelf-life while retaining most of the important growth factors and other molecules present in the fresh amniotic fluid for effective therapeutic application. Use of the compositions is intended for therapeutic purposes and will alleviate pain or discomfort associated with any disorders or diseases, particularly those involving eyes and joints, or fibrotic disorders such as COPD.