The establishment of human tumor cell lines has been a challenging endeavor, with many attempts failing due to the rarity of tumors with long-term in vitro growth potential and the need for specialized culture techniques. However, this chapter presents 31 new human tumor cell lines derived from 30 patients, demonstrating that successful establishment can be achieved through standard techniques and careful attention to detail. These cell lines originate from primary or metastatic tumors, including solid tumors and effusions. While many attempts have failed, the overall success rate suggests that various factors, including material selection, collection procedures, time between clinical procedures and tissue culture preparation, and technical competence, influence the outcome. The most critical factor appears to be the meticulous attention to detail by the tissue culturist. This chapter summarizes the methods used to collect tumor tissue, prepare for culture, and maintain the cell lines. It also reports on the growth characteristics, morphology, chromosome abnormalities, tumor-producing capacity, and ultrastructural features of the new cell lines, comparing them with previously established lines. It is noted that tumors of the same type can yield cell lines with different characteristics, highlighting the importance of using multiple cell lines for comprehensive cancer research. The chapter concludes with a discussion on the development of a large, well-characterized, contamination-free collection of human tumor cell lines, which is essential for various cancer research disciplines. The methods for collecting solid tumors and metastases are also described, emphasizing the importance of sterility and proper handling to ensure successful cell line establishment.The establishment of human tumor cell lines has been a challenging endeavor, with many attempts failing due to the rarity of tumors with long-term in vitro growth potential and the need for specialized culture techniques. However, this chapter presents 31 new human tumor cell lines derived from 30 patients, demonstrating that successful establishment can be achieved through standard techniques and careful attention to detail. These cell lines originate from primary or metastatic tumors, including solid tumors and effusions. While many attempts have failed, the overall success rate suggests that various factors, including material selection, collection procedures, time between clinical procedures and tissue culture preparation, and technical competence, influence the outcome. The most critical factor appears to be the meticulous attention to detail by the tissue culturist. This chapter summarizes the methods used to collect tumor tissue, prepare for culture, and maintain the cell lines. It also reports on the growth characteristics, morphology, chromosome abnormalities, tumor-producing capacity, and ultrastructural features of the new cell lines, comparing them with previously established lines. It is noted that tumors of the same type can yield cell lines with different characteristics, highlighting the importance of using multiple cell lines for comprehensive cancer research. The chapter concludes with a discussion on the development of a large, well-characterized, contamination-free collection of human tumor cell lines, which is essential for various cancer research disciplines. The methods for collecting solid tumors and metastases are also described, emphasizing the importance of sterility and proper handling to ensure successful cell line establishment.